Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription

Abstract

For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor–stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A₂₆₀ values (representing up to 40.8 μg RNA per 10⁶ cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low C_t values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.

Introduction

Human mesenchymal stem cells (hMSCs) are widely used in cell therapy and tissue engineering^1,2 due to their low in vitro and in vivo immunogenicity,^3,4 their ability to immunomodulate foreign cells,^1,5 and their lineage-overlapping differentiation capabilities. For many applications, hMSCs are embedded in hydrogels to achieve attachment, growth, and differentiation.⁶ The differentiation of hMSCs into particular tissues often requires three-dimensional cultivation in hydrogels as previously shown for the differentiation of stem cells into osteoblasts,⁷ chondrocytes,⁸ and nucleus pulposus cells.^9,10 Although synthetic hydrogel matrices are available, natural materials based on polysaccharides, such as alginate,¹¹ agarose,¹² hyaluronic acid,¹³ chitosan,¹⁴ and gellan gum,¹⁵ and/or proteins, such as gelatin,¹⁶ collagen,¹⁷ and fibrin,¹⁸ are often chosen because they resemble the in vivo environment of the differentiated target cells, thus stimulating hMSC differentiation in the required direction.

The differentiation of hMSCs is often monitored by quantitative reverse transcription-PCR (qRT-PCR), which requires the extraction of high-quality RNA. Many RNA extraction protocols for suspension cells are available, often in commercial kits whose basic principles involve RNA binding to silica membranes in spin columns in the presence of chaotropic salts or phenol/chloroform extraction, followed by RNA precipitation.¹⁹ However, the hydrogel material can interfere with RNA extraction because the positively charged polysaccharides and proteins form complexes with the negatively charged RNA and may also compete with the binding media. Any hydrogel components in the isolated RNA can hinder downstream applications such as qRT-PCR.

Plant-specific RNA extraction procedures have been used to address these challenges because the polysaccharide components of hydrogels are similar to those of plant tissues. For example, a plant-specific RNA extraction kit was used to isolate RNA from hMSCs embedded in agarose and gellan²⁰ and a plant-based CTAB (cetyltrimethylammonium bromide) method has been adapted to extract RNA from hMSCs embedded in chitosan, collagen, and agarose.²¹ The RNeasy extraction kit has been combined with the CTAB and Trizol protocols following the enzymatic homogenization of stem cells encapsulated in chitosan hydrogels.²² The relative merits of each of these methods are unclear and there has been no systematic comparison of different homogenization and RNA extraction methods applied to cells embedded in different hydrogel types.

We therefore compared a range of methods for the extraction of high-quality RNA from hMSCs embedded in different hydrogels, aiming to identify a procedure that is universally applicable regardless of the hydrogel type. We used three hydrogel types that are widely applied in tissue engineering experiments, that is, gelatin as a model protein-rich hydrogel, and agarose and alginate as models of polysaccharide-rich gels, each containing embedded cells from the cell line, hMSC-TERT. We compared four homogenization methods (three mechanical methods, plus combined chemical and enzymatic digestion specific for each hydrogel) combined with eight different RNA extraction protocols. The extracted RNA was evaluated in terms of yield (based on A₂₆₀) and purity (based on A₂₆₀/₂₈₀, A _260/230, and UV spectrum) and its suitability for endpoint and qRT-PCR.

Materials and Methods

Cultivation and harvesting of the hMSC-TERT line

The hMSC line, hMSC-TERT,²³ was expanded in cultivation medium as previously described^24,25 and passaged at 80% confluence. Cells with passage numbers 69–75 were used for RNA extraction.

Seeding cells into the hydrogels

Gelatin hydrogel

Gelatin hydrogels were seeded with 8 × 10⁵ cells/gel as previously described.⁹ The solidified hydrogel was transferred to a 12-well plate and overlaid with 1 mL cultivation medium. The cells were then incubated for 1 day at 37°C and 5% CO₂.

Agarose hydrogel

The hydrogel was prepared by dissolving low-gelling agarose (Applichem) in phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ (Merck Millipore) to a final concentration of 3% (w/v) and heating for 2 min in a microwave (800 W) before cooling to 42°C in a water bath. The cells were suspended in cultivation medium to a density of 1.2 × 10⁷ cells/mL, and 80 μL of the suspension was mixed with 160 μL of the cooled agarose solution by gentle pipetting. The hydrogel was prepared by transferring 200 μL of this mixture (8 × 10⁵ cells/gel) to a 24-well plate (TPP). The solidified hydrogel was then incubated as above.

Alginate hydrogel

The alginate hydrogel was prepared by dissolving alginate (Sigma-Aldrich Laborchemikalien GmbH) to a final concentration of 1.5% (w/v) in 0.9% (w/v) NaCl. The cells were suspended in cultivation medium at a density of 4 × 10⁷ cells/mL, and 24 μL of the suspension was mixed with 216 μL of the alginate solution before transferring 200 μL of this mixture to a 24-well plate. The hydrogel was formed by adding 1 mL 20 mM BaCl₂. After 1 h of incubation at room temperature, the BaCl₂ solution was removed and the hydrogel (8 × 10⁵ cells/gel) was washed twice with PBS (containing Ca²⁺ and Mg²⁺; Merck Millipore). The solidified hydrogel was treated as above.

Homogenization of seeded hydrogels

Microhomogenizer method

Before RNA extraction, the hydrogels were homogenized to maximize the RNA yield. For the microhomogenizer method, the cultivation medium was removed and each hydrogel was placed in a 1.5-mL RNase/DNase-free tube before mashing with a conical polypropylene microhomogenizer (Carl Roth) until all hydrogel structures were fragmented.

Rotor–stator method

The cultivation medium was removed and each hydrogel was placed in a 2-mL RNase/DNase-free tube to which we added 500 μL PBS (without Ca²⁺ and Mg²⁺). The hydrogel was shredded with a rotor–stator (RS) (Silentcrusher S; Heidolph) until a uniform viscous solution was obtained.

Liquid nitrogen method

Each hydrogel was placed in a mortar and covered with liquid nitrogen (LN). After the nitrogen had evaporated, the frozen hydrogel was pulverized using a pestle.

Enzymatic and chemical digestion

The cultivation medium was removed and each hydrogel was washed thrice in PBS (without Ca²⁺ and Mg²⁺). Gelatin hydrogels were supplemented with an enzyme mixture containing 200 μL 6.6 mg/mL proteinase K (Carl Roth) and 200 μL 10× trypsin (PAA Laboratories GmbH). Agarose hydrogels were placed in a 2-mL tube and heated to 70°C for 5 min to dissolve the hydrogel, before adding 200 μL 1× TBE buffer with 1 U agarase (Applichem). After initial digestion, each hydrogel was incubated for 24 h at 37°C to digest the gel matrix completely. Alginate hydrogels were dissolved by transferring each gel to a 15-mL Falcon tube containing 10 mL 10 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich) and 0.1 mg/mL bovine serum albumin (BSA; Carl Roth) in PBS (without Ca²⁺ and Mg²⁺) and vortexing for 20 min.

RNA extraction

Commercial kits and reagents

RNA was extracted using the AquaRNA™ kit (MoBiTec), Chemagic mRNA Direct Kit (PerkinElmer chemagen Technologie GmbH), Precellys Tissue RNA Kit (peqlab; VWR International GmbH), and RNeasy Mini Kit (Qiagen) according to the manufacturers’ recommendations. TRIzol^® Reagent (Qiagen) was used according to the manufacturer's recommendations, but at room temperature and with half the recommended volume. After extraction, RNasin^® Ribonuclease Inhibitor (Promega) was added to each isolated RNA sample to prevent degradation.

CTAB method

RNA was isolated in CTAB buffer²⁶ (2% [w/v] CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris, pH 8) containing 1% (v/v) β-mercaptoethanol. We briefly mixed 600 μL of the buffer with the homogenized hydrogel, added 600 μL chloroform, and centrifuged the mixture (14,000 g, 2 min, room temperature). The upper/aqueous phase was transferred to a fresh 2-mL tube for chloroform extraction. We then added 800 μL isopropanol and precipitated the RNA by centrifugation (14,000 g, 15 min, room temperature). The pellet was washed with 600 μL 70% (v/v) ethanol and dissolved in 90 μL RNase-free water for 15 min at 65°C. Impurities were removed by further centrifugation (14,000 g, 5 min, room temperature) and the supernatant was transferred to a fresh 2-mL tube. We added 30 μL 8 M LiCl (pH 8.0) and incubated the sample for 20 min at −20°C before further centrifugation (14,000 g, 30 min, 4°C). The supernatant was discarded and the pellet was washed with 100 μL 70% (v/v) ethanol and centrifuged (14,000 g, 2 min, room temperature). The final pellet was dissolved in 30 μL TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA) for 15 min at 65°C.

Hot phenol method

The homogenized hydrogel was vortexed for 30 s with 960 μL ice-cold cell resuspension buffer²⁷ (10 mM KCl, 10 mM MgCl₂, and 10 mM Tris-HCl, pH 7.5). The solution was split between two 2-mL tubes. We then added 400 μL hot phenol buffer (0.4 M NaCl, 26 mM EDTA, 1% β-mercaptoethanol, 35 mM sodium dodecylsulfate [SDS], 20 mM Tris-HCl, pH 7.5) and 80 μL phenol to each tube and vortexed the sample for 30 s. The mixture was incubated for 1 min at 95°C, followed by centrifugation (14,000 g, 15 min, room temperature). The supernatant was transferred to a fresh tube and the solution was mixed with 300 μL phenol and 300 μL chloroform and centrifuged (14,000 g, 2 min, room temperature). We transferred 500 μL of the upper clear phase to a fresh 2-mL tube, added 600 μL chloroform and 900 μL isopropanol, and incubated for 10 min on ice. After centrifugation (14,000 g, 15 min, room temperature), the pellet was washed with 1 mL 70% (v/v) ethanol, dried for 5 min, and dissolved in 30 μL TE buffer for 5 min at 37°C.

LiCl method

Each homogenized hydrogel was mixed with 500 μL 3 M LiCl and 500 μL 6 M urea and vortexed for 5 min.²⁸ The solution was centrifuged (4000 g, 15 min, room temperature) and incubated for 24 h at 4°C, followed by centrifugation (14,000 g, 20 min, 4°C). The supernatant was removed and the pellet was resuspended in 455 μL 10 mM Tris-HCl (pH 7.5) containing 70 mM SDS and 5 μL 20 mg/mL proteinase K and incubated for 30 min at 37°C. We then added 200 μL phenol and 200 μL chloroform, followed by vortexing and centrifugation (13,000 g, 5 min, room temperature). The aqueous phase was transferred to a fresh 2-mL tube, 400 μL chloroform was added, and the solution was vortexed, followed by centrifugation (13,000 g, 5 min, room temperature). The aqueous phase was transferred in a fresh 2-mL tube and the RNA was precipitated by mixing with 800 μL isopropanol and incubating for 30 min at −80°C, followed by centrifugation (13,000 g, 10 min, 4°C). The final pellet was washed with 1 mL 70% ethanol, followed by centrifugation (13,000 g, 5 min, 4°C), dried for 5 min, and dissolved in 30 μL TE buffer.

Determination of RNA yield

The RNA yield of each sample was calculated by measuring the absorbance at 260 nm (A₂₆₀), which corresponds to 40 μg/mL RNA per unit according to the Lambert–Beer law. We measured 2-μL samples using a Take3 plate (Biotek) and a Synergy plate reader (Biotek).

Determination of RNA purity

Absorbance spectra (220–350 nm) were acquired using a Take3 plate and a Synergy plate reader. The UV spectrum of each sample was compared with RNA extracted from pure cell suspension (positive control). The A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ ratios were calculated.

Endpoint RT-PCR

The suitability of each RNA sample for RT-PCR was determined by preparing cDNA using the Omniscript Reverse Transcription kit (Qiagen) according to the manufacturer's recommendations. Reverse transcription was carried out using the Taq PCR kit (Qiagen) according to the manufacturer's recommendations, with 1 ng RNA per reaction, oligo-dT primers (5′-TTT TTT TTT TTT TTT T-3′) provided by Eurofins MWG Operon. The RT-PCR was carried out using 50 ng cDNA. Primers for GAPDH⁹ (forward and reverse primer located in different exons [5 and 9] to confirm the absence of genomic DNA contamination) were used in a three-step RT-PCR method (initial denaturation for 3 min at 94°C, followed by 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 68°C, elongation for 1 min at 72°C, and a final elongation for 10 min at 72°C) using a Mastercycler (Eppendorf). The RT-PCR products (expected size: 835 bp) were analyzed by horizontal 2% (w/v) agarose gel electrophoresis (1 h, 70 V). The gels were stained with SYBR Gold (Thermo Fisher Scientific) and bands were detected under UV light.

Quantitative RT-PCR

The isolated RNA (2-μg aliquots) was transcribed into cDNA using the Precision nanoScript Reverse transcription kit with the oligo-dT primer included in the kit (Primerdesign Ltd), followed by qPCR (25 ng cDNA per reaction) using the Precision MasterMix premixed with SYBR Green (Primerdesign), according to the manufacturer's recommendations.²⁹ We used a Mastercycler realplex (Eppendorf) for a two-step qRT-PCR protocol (enzyme activation for 10 min at 95°C, followed by 50 cycles of denaturation for 15 s at 95°C, annealing/elongation for 1 min at 60°C), including primers for the detection of four housekeeping genes: EIF2A, encoding eukaryotic gene translation initiation factor 2A (primers provided by Primerdesign in the geNorm Kit); COL1A, encoding collagen type 1 (forward primer 5′-GGG CCT CAG GGT GCT CGA GGA TTC-3′, reverse primer 5′-AGG GCT GCC AGG GCT TCC AGT CAG A-3′); IBSP, encoding integrin-binding sialoprotein (forward primer 5′-GGG CAC CTC GAA GAC ACA ACC TC-3′, reverse primer 5′-TCC CCC TCG TAT TCA ACG GTG GTG-3′); and PAX1, encoding paired box protein 1 (forward primer 5′-TTC AAG CAT CCC AGC CGA GAA GGA-3′, reverse primer 5′-AGT CCG TGT AAG CTA CTG AGG GCG-3′). The correct product sizes were confirmed by melting temperature (T_m) analysis in the range 60–95°C: EIF2A = 113 bp (T_m measured = 83°C); COL1A = 139 bp (T_m predicted = 85°C, T_m measured = 90°C); IBSP = 120 bp (T_m predicted = 84°C, T_m measured = 82°C); and PAX1 = 79 bp (T_m predicted = 81°C, T_m measured = 85°C).

Statistical analysis

Each combination of homogenization and RNA extraction methods was applied to three independent samples for each hydrogel type (three sample replicates). The RNA yield and purity were determined thrice, and the RT-PCR experiments were also carried out thrice (three measurement replicates). The three measurement replicates were used to calculate the mean and standard deviation for each sample, whereas the mean and standard deviation for each RNA extraction method were calculated using the sample replicates. To compare the mean values, a t-test (two-sample test with known variances) was performed. A p-value under 0.05 was deemed to indicate a statistically significant difference.

Results

RNA yields

The hMSC-TERTs were embedded in three different hydrogel matrices (alginate, agarose, and gelatin). The cell–matrix composites were homogenized using four different methods and RNA was extracted using eight different procedures. The extracted RNA was then evaluated in terms of yield and purity. We used RNA extracted from an hMSC-TERT suspension culture as a control to estimate the RNA yield and purity when the extraction was not influenced by the hydrogel matrix.

Figure 1 shows the total RNA yields (based on A₂₆₀ values) achieved using each of the different procedures. Depending on the homogenization method, enzymatic/chemical digestion (ED) and microhomogenization (MH) produced the highest quantity of RNA for all matrices. For agarose matrices, hydrogel homogenization by MH was more efficient than ED, whereas for alginate and gelatin, there was no difference. Homogenization with LN was unsuitable for the gelatin and alginate hydrogels, but effective for alginate hydrogels. Homogenization with the RS appeared to be unsuitable for all matrices because little or no RNA could be detected after extraction.

FIG. 1.

Total RNA yield from hMSC-TERTs embedded in different hydrogel matrices based on different homogenization and RNA extraction methods. The RNA yield of the samples was calculated according to the Lambert–Beer law based on absorbance at 260 nm (1 A₂₆₀ unit = 40 μg/mL RNA). Three independent samples were each measured in triplicates. Standard deviation is given in the Supplementary Data; Supplementary Data are available online at www.liebertpub.com/tec. As control, RNA extracted from nonembedded hMSC-TERTs is shown. The mean values were compared using a t-test (two-sample test with known variances). The p-value was under 0.05. hMSC-TERT, human mesenchymal stem cell telomerase reverse transcriptase; Homogenization methods: ED, enzymatic/chemical digestion; LN, liquid nitrogen; MH, microhomogenization; RS, rotor–stator. RNA extraction protocols: AQ, Aqua RNA; CM, Chemagic; CT, CTAB; HP, hot phenol; LC, LiCl; PC, Precelly; RE, RNeasy; TR, Trizol.

We achieved remarkable RNA yields using the hot phenol (HP), Trizol (TR), and LiCl (LC) protocols with average yields of 8.1 ± 14.3 (HP), 6.1 ± 5.1 (TR), and 2.8 ± 2.0 (LC) μg per 10⁶ hMSC-TERT cells embedded in each of the three hydrogel types. The Aqua RNA (AQ), CTAB (CT), and Precelly (PC) protocols produced moderate yields of 1.6 ± 1.2 (AQ), 1.5 ± 2.0 (CT), and 0.5 ± 0.5 (PC) μg per 10⁶ embedded cells, whereas the poorest yields, typically below the detection limit, were observed with the RNeasy (RE) and Chemagic (CM) procedures. On average, the best-performing RNA extraction procedure achieved similar yields to the mean of the pure cell control (7.6 ± 6.2 μg per 10⁶ cells, p < 0.05). A₂₆₀ values reporting RNA yields that significantly exceeded the pure cell control indicated the presence of absorbance-increasing impurities in the sample.

None of the RNA extraction protocols were ideal for all hydrogel matrices and all homogenization methods because each protocol led to RNA yields below the detection limit in at least one case. Among the 12 combinations of procedures, the failure rate was between 1/12 for LC and 10/12 for RE and CM. Regardless of the choice of hydrogel, the LC RNA extraction protocol offered the best chance to recover sufficient yields of RNA from embedded hMSC-TERT cells.

RNA purity

RNA purity was assessed by measuring the absorbance ratios at 260/280 nm and 260/230 nm, with pure samples achieving an A₂₆₀/A₂₈₀ ratio between 1.9 and 2.1, and an A₂₆₀/A₂₃₀ ratio ≥2. The RNA quality was evaluated by comparing the UV spectra of the samples with that of the pure RNA control, the latter showing a smooth curve with a clear maximum at 260 nm and low absorbance values below 230 nm and above 300 nm (Fig. 2A). Any changes in the shape, for example, increasing values around 220–230 nm or any shoulder formation (Fig. 2B), were indications of sample contamination.³⁰

FIG. 2.

UV spectra representing RNA samples varying in quality. (A) Pure RNA control (e.g., hMSC-TERT without hydrogel, RNA extracted with RNeasy kit). (B) Low-level contamination (e.g., hMSC-TERT embedded in alginate, homogenized with microhomogenization, and RNA extracted with the Aqua RNA kit). (C) High-level contamination (e.g., hMSC-TERT embedded in agarose, homogenized with enzymatic/chemical digestion, and RNA extracted with hot phenol protocol). The UV spectra span the range 220–350 nm and were acquired using a plate reader.

Table 1 shows the A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ ratios and quality evaluations for the UV spectra representing each sample. Most of the extraction protocols produced impure RNA, including the best-performing RNA extraction protocol (HP). Only the CT extraction method, which was moderate in terms of yield, achieved A₂₆₀/A₂₈₀ ratios between 1.9 and 2.1 for all three hydrogel types and for more than one of the homogenization methods. Similarly, the A₂₆₀/A₂₃₀ ratio was ≥2 in most cases and the UV spectra were similar to that of the control. The HP, AQ, and RE extraction methods achieved A₂₆₀/A₂₈₀ ratios between 1.9 and 2.1 on one occasion each, but the UV spectra were poor as shown for the RNA isolated from hMSC-TERTs embedded in agarose and extracted using the ED and HP methods (Fig. 2C). The RNA quality analysis thus contrasted with the RNA yield data. The CT method, which only produced moderate RNA yields, achieved the purest RNA.

Table 1.

A_260/280 and A_260/230 Ratios and UV Spectra Representing All Isolated RNA Samples

	Agarose				Alginate				Gelatin
	RS	LN	MH	ED	RS	LN	MH	ED	RS	LN	MH	ED	Control
HP
A_260/280	1.3 ± 0.1	OOR	1.4 ± 0.3	1.9 ± 0.0	1.0 ± 0.1	1.2 ± 0.1	1.2 ± 0.2	1.0 ± 0.2	OOR	OOR	1.5 ± 0.0	1.4 ± 0.0	1.4 ± 0.4
A_260/230	1.4 ± 0.2	OOR	0.5 ± 0.2	0.9 ± 0.2	0.6 ± 0.1	0.9 ± 0.2	0.7 ± 0.2	0.3 ± 0.0	OOR	OOR	2.1 ± 0.0	1.7 ± 0.1	1.9 ± 0.2
UV spectrum	−	−	−	−	−	−	−	−	−	−	−	−	+
TR
A_260/280	1.2 ± 0.1	1.2 ± 0.0	1.3 ± 0.1	OOR	1.5 ± 0.1	1.1 ± 0.8	1.5 ± 0.1	1.3 ± 0.1	OOR	1.7 ± 0.1	1.6 ± 0.1	1.6 ± 0.1	1.9 ± 0.2
A_260/230	0.3 ± 0.0	0.2 ± 0.0	0.2 ± 0.2	OOR	0.4 ± 0.0	0.3 ± 0.2	0.4 ± 0.0	0.4 ± 0.2	OOR	0.6 ± 0.2	0.3 ± 0.1	0.5 ± 0.3	1.9 ± 0.1
UV spectrum	−	−	−	−	−	−	−	−	−	−	−	−	+
LC
A_260/280	1.4 ± 0.0	1.4 ± 0.0	1.5 ± 0.0	1.4 ± 0.0	1.5 ± 0.0	1.5 ± 0.0	1.5 ± 0.0	1.4 ± 0.0	1.5 ± 0.0	1.4 ± 0.0	1.4 ± 0.1	OOR	1.8 ± 0.2
A_260/230	1.5 ± 0.1	1.4 ± 0.1	1.8 ± 0.0	1.8 ± 0.1	1.7 ± 0.1	0.7 ± 0.2	1.9 ± 0.0	1.5 ± 0.2	1.1 ± 0.1	1.7 ± 0.1	0.9 ± 0.2	OOR	2.0 ± 0.1
UV spectrum	−	−	−	−	−	−	−	−	−	−	−	−	+
CT
A_260/280	OOR	OOR	2.0 ± 0.0	2.2 ± 0.1	OOR	2.0 ± 0.1	2.0 ± 0.0	1.9 ± 0.2	OOR	2.0 ± 0.0	1.9 ± 0.0	2.0 ± 0.8	1.9 ± 0.2
A_260/230	OOR	OOR	1.9 ± 0.5	1.0 ± 0.1	OOR	OOR	2.1 ± 1.2	1.9 ± 0.6	OOR	OOR	2.3 ± 1.1	1.1 ± 0.8	2.0 ± 0.1
UV spectrum	−	−	+	+	−	−	+	+	−	+	+	+	+
AQ
A_260/280	1.7 ± 0.1	0.9 ± 1.8	1.6 ± 0.2	1.6 ± 0.2	1.4 ± 0.8	1.5 ± 0.1	1.8 ± 0.3	1.7 ± 0.1	OOR	1.8 ± 0.0	OOR	OOR	1.9 ± 0.3
A_260/230	0.1 ± 0.1	0.1 ± 0.2	0.0 ± 0.0	0.2 ± 0.2	0.1 ± 0.2	0.4 ± 0.1	0.1 ± 0.0	0.5 ± 0.0	OOR	0.6 ± 0.2	OOR	OOR	2.1 ± 0.1
UV spectrum	−	−	−	−	−	+	−	+	−	+	−	−	+
PC
A_260/280	OOR	OOR	OOR	OOR	OOR	1.2 ± 0.3	1.9 ± 0.1	1.4 ± 0.2	OOR	1.6 ± 0.2	0.7 ± 0.0	1.8 ± 0.1	2.0 ± 0.0
A_260/230	OOR	OOR	OOR	OOR	OOR	0.2 ± 0.1	0.1 ± 0.1	0.2 ± 0.1	OOR	0.3 ± 0.1	1.6 ± 0.2	0.4 ± 0.1	2.0 ± 0.2
UV spectrum	−	−	−	−	−	−	−	+	−	+	−	+	+
RE
A_260/280	OOR	OOR	OOR	OOR	OOR	1.0 ± 0.3	OOR	OOR	OOR	OOR	OOR	1.7 ± 0.3	2.0 ± 0.1
A_260/230	OOR	OOR	OOR	OOR	OOR	0.1 ± 0.0	OOR	OOR	OOR	OOR	OOR	0.2 ± 0.2	2.1 ± 0.0
UV spectrum	−	−	−	−	−	−	−	−	−	−	−	+	+
CM
A_260/280	OOR	OOR	1.4 ± 0.2	OOR	1.1 ± 0.3	OOR	1.0 ± 0.3	OOR	OOR	OOR	OOR	OOR	OOR
A_260/230	OOR	OOR	0.0 ± 0.0	OOR	1.4 ± 0.6	OOR	1.0 ± 0.5	OOR	OOR	OOR	OOR	OOR	OOR
UV spectrum	−	−	−	−	−	−	−	−	−	−	−	−

The A_260/280 ratio is 1.9–2.1 and the A_260/230 ratio is ∼2 for pure RNA. If one absorbance value was below the detection limit (0.01), no reliable ratio could be calculated (OOR = out of range). The quality of each UV spectrum quality was defined as (+) when it was identical to the pure RNA control spectrum (Fig. 2A) or as (−) when high values were observed in the 220–230 nm region and the maximum absorbance peak was shifted to a higher wavelength than 260 nm or a shoulder was observed (Fig. 2B) or the spectral shape was completely disrupted (Fig. 2C). Each measurement was taken in triplicate. As control, RNA extracted from nonembedded hMSC-TERTs is shown. OOR—out of range, values not given as at least one absorbance were too low for reliable ratios.

Homogenization methods: ED, enzymatic/chemical digestion; LN, liquid nitrogen; MH, microhomogenization; RS, rotor–stator. RNA extraction protocols: AQ, Aqua RNA; CM, Chemagic; CT, CTAB; HP, hot phenol; LC, LiCl; PC, Precelly; RE, RNeasy; TR, Trizol.

Suitability for endpoint RT-PCR

Each RNA sample was used as a template for RT-PCR, and the products were analyzed by horizontal agarose gel electrophoresis. The results were deemed positive when the anticipated product size was correct and there were no additional bands. Approximately 80% of the isolated RNA samples generated the anticipated products (Table 2). RNA samples isolated with the favored CT method yielded correct RT-PCR products when using the LN, MH, and ED homogenization methods, but not the RS method. Correct RT-PCR products were generated in some cases even when the RNA yield in the corresponding sample was below the detection limit of UV spectrophotometry, for example, from samples isolated using the RE protocol.

Table 2.

Endpoint Reverse Transcription–Polymerase Chain Reaction Results Using Isolated RNA Samples as Templates

	Agarose				Alginate				Gelatin
	RS	LN	MH	ED	RS	LN	MH	ED	RS	LN	MH	ED
HP	+	+	+	+	+	+	+	+	+	+	+	+
TR	+	+	+	+	−	+	−	−	+	+	+	+
LC	−	+	−	−	+	+	−	+	−	+	−	+
AQ	+	+	+	−	+	+	+	+	+	+	+	−
CT	−	+	+	+	−	+	+	+	−	+	+	+
CM	+	+	+	+	+	+	+	+	+	+	+	+
PC	+	+	+	+	−	−	−	−	+	+	+	+
RE	+	+	+	+	+	+	+	+	+	+	+	+

+ = positive result (correct product size, no additional band); − = negative result (dominant additional band, PCR product with incorrect size, or no PCR product). Each experiment was carried out thrice.

Suitability for qRT-PCR

The CT RNA extraction protocol was preferable for hMSC-TERTs embedded in each of the three hydrogel types. The suitability of the isolated RNA for qRT-PCR was therefore determined only for these samples. RNA samples isolated using the TR protocol were used as a control because they achieved not only high RNA yields but also substantial quantities of impurities, which nevertheless generated the correct RT-PCR products.

The quality of RNA in a qRT-PCR experiment can be determined by analyzing the cycle threshold (C_t) and the melting curves. High C_t values indicate low-quality RNA, whereas low C_t values (≤20) indicate high-quality RNA. Four different primer sets produced C_t values of 22.6–24.5 for the CT samples and 27.6–44.5 for the TR samples. Figure 3 shows representative C_t values for RNA samples isolated using a combination of MH and the TR or CT protocols. The melting temperatures of the qRT-PCR products were correct and thereby anticipated the product sizes for each reaction.

FIG. 3.

Quantitative RT-PCR using isolated RNA samples as template. Agarose-embedded hMSC-TERTs were disrupted by microhomogenization, followed by the CTAB (CT) or Trizol (TR) RNA extraction protocols. The RNA samples were used as qRT-PCR templates for the amplification of four genes expressed in hMSC-TERTs. The cycle threshold (C_t) values of three independent samples were measured in triplicate. Standard deviations are given as error bars. qRT-PCR, quantitative reverse transcription-PCR.

Discussion

The extraction of high-quality RNA from cells embedded in hydrogels is a fundamental requirement for the analysis of any tissue engineering approach by endpoint or quantitative RT-PCR. However, this remains a challenge because hydrogel materials interfere with most RNA extraction methods.

We systematically analyzed different procedures for the extraction of RNA from embedded hMSC-TERTs. Substantial differences in RNA quantity and quality were observed when different homogenization methods were compared. RS and LN produced poor RNA yields, in the former case, because harsh shear forces can damage the RNA and, in the latter case, because LN increases exposure to endogenous RNases.³¹ The ED homogenization methods achieved complete dissolution of the hydrogel matrix and the cells were released into suspension, resulting in much higher RNA yields. The MH method also produced high RNA yields, suggesting that there was little damage to the RNA during homogenization.

All precipitation-based methods (HP, TR, LC, AQ, and CT) produced high or moderate RNA yields from the embedded hMSC-TERT material. Precipitation can improve RNA extraction from plant tissues by removing interfering contaminants.³² The precipitation-based methods used basic pH conditions, which discourage the formation of complexes between the RNA and the hydrogel components, thus leading to higher RNA yields.²¹ We found that extraction methods based on selective RNA binding (PC, RE, and CM) were less suitable for hydrogel applications because the hydrogel components reduce the efficiency of RNA capture.³³ In previous studies, maximum RNA yields of 4.5 and 9.2 μg per 10⁶ cells have been reported,^21,22 thus our maximum of 40.8 μg per 10⁶ cells appeared comparatively high. However, the analysis of these samples revealed the presence of impurities that may increase apparent yields based solely on A₂₆₀ readings.

We found that ED and MH produced high-quality RNA consistently, LN sometimes, but RS never, suggesting the latter was too harsh for efficient RNA recovery. The variable performance of LN during the preparation of RNA from embedded hMSCs has been noted previously, with some achieving high yields of good quality RNA²¹ and others reporting poor yields and low quality.²²

The RNA extraction methods based on selective binding produced poor yields and low quality, with the UV spectra indicating significant contamination from hydrogel components. RNA samples isolated using the HP, TR, and LC protocols achieved high A₂₆₀ values, but the UV spectra indicated substantial contamination, which would exaggerate the reported yields. Absorbance values provide only indicative assessments and full UV spectra are needed to reveal the presence of contamination.²² We found that only the RNA samples isolated using the CT protocol achieved A_260/280 and A_260/230 ratios similar to the control combined with UV spectra confirming the absence of impurities. The ability of the CT protocol to produce high-quality RNA from hydrogel-embedded cells may reflect the combination of CTAB and β-mercaptoethanol under high salt and basic pH conditions. The high pH inhibits the formation of complexes between the hydrogel matrix and RNA²¹ and the strong detergent CTAB helps to lyse the cells and separate the RNA from polysaccharides/proteins.³⁴ The β-mercaptoethanol inactivates endogenous RNases. Additionally, the high salt content promotes the solubilization of polysaccharide/protein aggregates and the CTAB-RNA complexes, resulting in the efficient removal of hydrogel components and CTAB during chloroform extraction.³⁵

RNA samples with appropriate A_260/280 ratios and UV spectra can nevertheless produce incorrect RT-PCR products or high C_t values in qRT-PCR experiments. We found that RNA isolated from alginate hydrogels by RS, followed by the CT protocol, had an A_260/280 ratio of 1.9 and a well-shaped UV spectrum, but was unsuitable as an RT-PCR template. This agrees with previous reports showing that high yields of RNA with A_260/280 ratios within the expected range can be unsuitable for downstream analysis.²² In contrast, we achieved positive endpoint RT-PCR results with impure RNA, for example, following extraction using the TR protocol. The qRT-PCR experiments confirmed that these RNA samples were unsuitable for further analysis due to their high C_t values of up to 45, although further purification methods could be used to remove residual contaminants from such samples. The positive outcome of the ED and MH methods, followed by the CT extraction protocol, was confirmed in the endpoint and qRT-PCR experiments, which achieved C_t values of 22–24. This was in good agreement with previous studies reporting C_t values of 20–25 for RNA isolated from chitosan-embedded hMSCs.²¹

We identified a universal RNA extraction procedure that was satisfactory regardless of the hydrogel type. The major challenges were to homogenize the cells and the hydrogel matrix effectively without damaging the RNA and to extract the RNA successfully from an environment rich in polysaccharides and proteins. Our recommended procedure comprises MH or enzymatic/chemical digestion, followed by RNA precipitation in the presence of the RNA-chelating agent, CTAB. This procedure should also be transferable to other cell types embedded in the hydrogel types we investigated.

RNA extraction is often seen as a necessary evil, but it has an enormous impact on subsequent analysis. We strongly recommend the critical evaluation of RNA extraction methods before commencing tissue engineering experiments with embedded cells. An optimized RNA extraction procedure produces high yields of good quality RNA that is suitable for sensitive downstream analysis methods.

Footnotes

Acknowledgments

The authors acknowledge the Hessen State Ministry of Higher Education, Research and the Arts for financial support within the initiative, Putting research into practice, and the Hessen initiative for scientific and economic excellence (LOEWE program). The authors would like to thank Dr. Richard M. Twyman for assistance with writing and editing.

Disclosure Statement

No competing financial interests exist.

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Supplementary Material

Please find the following supplemental material available below.

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	Agarose				Alginate				Gelatin
	RS	LN	MH	ED	RS	LN	MH	ED	RS	LN	MH	ED
HP	+	+	+	+	+	+	+	+	+	+	+	+
TR	+	+	+	+	−	+	−	−	+	+	+	+
LC	−	+	−	−	+	+	−	+	−	+	−	+
AQ	+	+	+	−	+	+	+	+	+	+	+	−
CT	−	+	+	+	−	+	+	+	−	+	+	+
CM	+	+	+	+	+	+	+	+	+	+	+	+
PC	+	+	+	+	−	−	−	−	+	+	+	+
RE	+	+	+	+	+	+	+	+	+	+	+	+

	Agarose				Alginate				Gelatin
	RS	LN	MH	ED	RS	LN	MH	ED	RS	LN	MH	ED
HP	+	+	+	+	+	+	+	+	+	+	+	+
TR	+	+	+	+	−	+	−	−	+	+	+	+
LC	−	+	−	−	+	+	−	+	−	+	−	+
AQ	+	+	+	−	+	+	+	+	+	+	+	−
CT	−	+	+	+	−	+	+	+	−	+	+	+
CM	+	+	+	+	+	+	+	+	+	+	+	+
PC	+	+	+	+	−	−	−	−	+	+	+	+
RE	+	+	+	+	+	+	+	+	+	+	+	+

Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription–Polymerase Chain Reaction Analysis

Abstract

Introduction

Materials and Methods

Cultivation and harvesting of the hMSC-TERT line

Seeding cells into the hydrogels

Gelatin hydrogel

Agarose hydrogel

Alginate hydrogel

Homogenization of seeded hydrogels

Microhomogenizer method

Rotor–stator method

Liquid nitrogen method

Enzymatic and chemical digestion

RNA extraction

Commercial kits and reagents

CTAB method

Hot phenol method

LiCl method

Determination of RNA yield

Determination of RNA purity

Endpoint RT-PCR

Quantitative RT-PCR

Statistical analysis

Results

RNA yields

RNA purity

Suitability for endpoint RT-PCR

Suitability for qRT-PCR

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Supplementary Material

	Agarose				Alginate				Gelatin
	RS	LN	MH	ED	RS	LN	MH	ED	RS	LN	MH	ED
HP	+	+	+	+	+	+	+	+	+	+	+	+
TR	+	+	+	+	−	+	−	−	+	+	+	+
LC	−	+	−	−	+	+	−	+	−	+	−	+
AQ	+	+	+	−	+	+	+	+	+	+	+	−
CT	−	+	+	+	−	+	+	+	−	+	+	+
CM	+	+	+	+	+	+	+	+	+	+	+	+
PC	+	+	+	+	−	−	−	−	+	+	+	+
RE	+	+	+	+	+	+	+	+	+	+	+	+