Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization

The isolation of high‐quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate‐phenol‐chloroform extraction method, which involves enzymatic predigestion of the tissue prior to standard RNA isolation. Yield, purity and integrity of RNA isolated from bovine nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were compared among complete matrix digestion, predigestion and pulverization, pulverization alone, and pulverization followed by on‐column purification. With predigestion, the average yield of RNA obtained from bovine nucleus pulposus was 8.82 ± 2.05 ng/mg of wet tissue with 260/280 and 260/230 optical density ratios of 1.91 ± 0.15 and 1.84 ± 0.30, respectively. RIN analysis indicated that RNA quality was best preserved with the predigestion method (RNA integrity number > 7), and the extracted RNA was suitable for real‐time polymerase chain reaction. This method is of importance for gene expression studies on intervertebral disc development, degeneration and repair, and we anticipate that it may be further applied to other tissues rich in proteoglycans.


| INTRODUCTION
The intervertebral disc (IVD) is composed of an outer fibrotic tissue rich in type I collagen (annulus fibrosus, AF) and a central jelly core rich in proteoglycans and type II collagen (nucleus pulposus, NP). The AF itself can be separated in outer and inner AF, with the latter being a transition zone between the outer AF and the central NP. While there is a natural evolution of the IVD macroscopic structure and phenotype during life, IVD degeneration is considered to be an accelerated aging process of the IVD and has often been associated to low back pain. 1 Since the repair capacity of the IVD is limited, tissue engineering strategies for IVD repair have been extensively investigated and hydrogels have been suggested for NP repair, while fibrous structures have proven to be beneficial for AF restoration. 2 Most recent strategies aim at rejuvenating the aging disc by the use of stem cells 3,4 and at enhancing the endogenous repair of the disc by injecting an appropriate stimulating factor. 5 Gene expression analysis plays an important role in the assessment of the success of IVD repair strategies as it can provide useful information about cellular mechanisms of tissue repair and restoration of a healthy cell phenotype. Besides, such analyses are also used to investigate the phenotype of the IVD cells, including transcriptome studies. Gene expression analyses rely on the isolation of high-purity RNA from disc tissue. However, the isolation of RNA from the IVD, and especially from the central jelly-like NP tissue, is particularly challenging due to a unique combination of low cellularity (~4000 cells/mm 3 ) and high proteoglycan content. 1 One notable proteoglycan, aggrecan, tends to coprecipitate with RNA during the RNA isolation process. 6 Considering the high amounts of proteoglycans present in cartilage, several protocols have been specifically developed for the successful isolation of RNA from cartilage. [7][8][9][10] There have been other techniques described for the isolation of RNA from disc tissue which can avoid the problem of proteoglycan coprecipitation, such as enzymatic tissue digestion, 11 although the yields from single discs are relatively low. Additionally, specific protocols have been developed for the isolation of RNA from cells cultured in polysaccharide-based hydrogels. 12,13 However, these techniques may not be as successful when applied to disc tissue, which has an even higher proteoglycan content and lower cellularity. Recently, Lee et al have proposed cryosectioning, second phase separation and high salt precipitation to obtain RNA suitable for real-time polymerase chain reaction (RT-PCR) from IVD tissues. 14 In another study, Peeters et al have suggested to disrupt and homogenize the tissue using rapid agitation of ceramic balls in the presence of the lysis buffer, followed by proteinase K digestion and purification with a silica column. 15 However, the 260/230 ratio obtained by these methods is relatively low, suggesting the presence of contaminants (proteoglycans, extraction buffer) or degraded RNA.
Our goal was to develop a method for isolating high-quality RNA from all disc tissues that allows using small amounts of tissues (approximately 100 mg per sample). Indeed, the amount of tissue available can be limited when caudal discs from large animals or IVDs from small animals (eg, mouse, rat, rabbit) are used. In addition, the number of samples to process from a single experiment can be relatively high when NP, inner AF and outer AF are collected from multiple discs. In this study, we compare 3 previously established methods (complete tissue digestion, pulverization only, pulverization and on-column purification) for the isolation of RNA from bovine disc tissue to a new protocol (predigestion and pulverization) that produces reliable amounts of high-quality RNA. This new method is based on predigestion of the tissue with pronase prior to extraction with guanidinium thiocyanate-phenol-chloroform. We demonstrate that this method is valid for the isolation of RNA from all tissues of the IVD, namely NP, inner and outer AF.

| Intervertebral disc samples
IVDs comprising cartilaginous endplates were harvested from bovine tails (10-12 months old) obtained from a local abattoir within 2 hours of death and washed in phosphate buffered saline (PBS) containing 10% penicillin-streptomycin (Gibco, Zug, Switzerland) for 10 minutes, followed by a second wash in PBS with 1% penicillin-streptomycin.
Three bovine tails were dissected, and the 2 biggest discs were collected from each tail. IVD health state was confirmed by visual inspection by an experienced scientist. Thickness of AF (normal or not), jelly behavior of NP (quick shape recovery following deformation induced with a spatula) and absence of vasculature ingrowth inside the IVD were assessed. Discs were cultured overnight in Dulbecco's modified Eagle's medium (DMEM 4.5 g/L glucose) with 2% fetal bovine serum (FBS, Gibco), 1% insulin transferrin selenium and 0.2% Primocin (Invivogen, Nunnigen, Switzerland). From each disc, NP, inner and outer AF tissues were collected and a piece (100-150 mg) of each tissue type was randomly assigned to 1 of the 4 RNA isolation groups.
Tissue from each disc and tail was treated separately (n = 6). For RNA isolation, NP and inner and outer AF tissues from each disc were separated and chopped into small pieces (1−3 mm 3 ) using a scalpel. The minced tissues were stored in 5 mL PBS/sample to avoid tissue drying until all samples were prepared. The tubes containing the minced tissues were briefly centrifuged (500g, 2 minutes) and PBS was aspirated.

| Complete matrix digestion method
Minced tissue samples (n = 6 per tissue type) were digested using a two-step enzymatic digestion (pronase followed by collagenase), as previously described. 11 Briefly, tissue was digested at 37 C on a wav-

| Predigestion and pulverization method
Minced tissue samples (n = 6 per tissue type) were digested at 37 C for 1 hour in DMEM with pronase using the same amounts and concentrations as specified above. The digestion was then stopped with FBS and the tissue was washed twice with PBS. Any remaining PBS was aspirated, and tissue was snap-frozen and pulverized in liquid nitrogen with a custom-made pestle device ( Figure 1). Pulverization was performed in the frozen state in the presence of liquid nitrogen and lasted few seconds. Powdered samples were transferred in the frozen state to 2 mL microcentrifuge tubes containing 1 mL TRI Reagent and 5 μL of polyacryl carrier and homogenized using a Tissue Lyzer (Qiagen, Switzerland) at 25 Hz for 6 minutes with a single 8 mm diameter stainless steel ball. Following centrifugation at 2000g for 2 minutes to remove remaining tissue debris, the supernatant was transferred to a 1.5 mL microcentrifuge tube and RNA isolation continued as described above.

| Pulverization only method
Minced tissue samples (n = 6 per tissue type) were frozen and pulverized in liquid nitrogen with a custom-made pestle device. The resulting powder was mixed with 1 mL TRI Reagent and 5 μL of polyacryl carrier and homogenized using a Tissue Lyzer as described above. Following centrifugation at 2000g for 2 minutes to remove remaining tissue debris, the supernatant was transferred to a 1.5 mL microcentrifuge tube and RNA isolation continued as described above.

| Pulverization and on-column purification method
Minced tissue samples (n = 6 per tissue type) were frozen and pulverized in liquid nitrogen with a custom-made pestle device. The resulting powder was mixed with 1 mL TRI Reagent and 5 μL of polyacryl carrier and homogenized using a Tissue Lyzer. Following centrifugation for at 2000g 2 minutes to remove remaining tissue, the supernatant was transferred to a new 1.5 mL tube. RNA isolation continued as described above until phase separation: the resulting aqueous layer was mixed with pure ethanol (0.7 mL ethanol per 1 mL of acqueous phase), transferred to an RNeasy column (Qiagen) and cleaned according to manufacturer's instructions. RNA was eluted with 30 μL of RNase-free water.

| RNA integrity number analysis
To complete the results obtained spectrophotometrically, a RNA integrity number (RIN) evaluation was conducted on a separate set of samples (n = 2 per group). The RNA concentration and purity (260/280 ratio) of the samples were determined spectrophotometrically and samples were sent to Microsynth AG (Balgach, Switzerland) for RIN analysis. Samples were quantitatively assessed using an Agilent 2100 Bioanalyzer System and Agilent 2100 expert software (Agilent Technologies, Santa Clara, CA, USA). 17 The Bioanalyzer software provided the ratio of the 18S to 28S ribosomal subunits by applying an algorithm to the whole electropherogram. The RIN was used as an indicator of RNA quality (scale from 1 to 10, with 1 strongly degraded RNA and 10 intact RNA).

| Statistical analysis
Since the tissues (NP, inner AF, outer AF) collected from 1 disc were split into 4 groups (the 4 RNA isolation methods) and 6 discs were used in the study, a two-way analysis of variance (ANOVA) with subject matching and Tukey post-hoc was performed (P < .05 was considered significant) for RNA yield, concentration, 260/280 and 260/230 ratios. A one-way ANOVA with subject matching and Tukey post-doc (P < .05 was considered significant) was performed on the Bioanalyzer data and corresponding Nanodrop data (data from different tissues using the same RNA isolation method were combined in one group). For gene expression analyses, since not all genes of interest were detected in all samples, a two-way ANOVA without subject matching was performed with Tukey post-hoc (P < .05 was considered significant).

| RESULTS
The yield, concentration and purity of the RNA obtained with the 4 RNA isolation techniques are summarized in Figure 2 (and Table S1, . Data are represented as mean AE SD; n = 3 to 6. Red dotted line represents the suggested acceptable threshold minimum value for each parameter. Statistical analysis of the RNA extraction method for each tissue type: "a" vs complete matrix digestion, "b" vs predigestion and pulverization, "c" vs pulverization only, and "d" vs pulverization and on-column; 0 <.05, 00 <.01, 000 <.001. Statistically significant differences among tissues processed with the same RNA extraction method are represented with a line; * <.05, ** <.01, *** <.001 obtained with predigestion and pulverization, followed by complete matrix digestion, while pulverization (alone or in combination with oncolumn purification) gave the lowest RNA concentrations (<15 ng/μL).  (Figure 3 and Table S2). Pulverization resulted in strong RNA degradation, which could only partially be recovered by further purification with a column (RIN < 3 for NP and RIN < 5 for AF). Interestingly, the predigestion and pulverization resulted in the lowest variability in RNA concentrations for all tissue types, highest RNA concentration (measured by Bioanalyzer) with lowest variability among replicas, and highest 260/280 ratios ( Figure 3 and Table S2) TRIzol method compared to isolation with TRIzol followed by purification of the aqueous phase using a column-based method. 14 Of note, RNA yield was very low using the RNeasy column (cell lysis in RLT buffer provided in the Qiagen RNeasy kits followed by on-column purification). In our hands (data not shown), phenol-guanidine isothiocyanate-based solutions are superior for lysis of NP tissue compared to RLT buffers (Qiagen RNeasy kits) and cetyl trimethylammonium bromide. 12 Interestingly, the RNA extraction method suggested by Peeters et al involves an enzymatic digestion step with proteinase K after the homogenization and it was suggested in this study that a higher volume of enzymatic solution improves both the yield and quality of the extracted RNA. 15 This supports our finding that a short enzymatic digestion is an important step when isolating RNA from IVD tissues. In our hands, this step is most beneficial when performed prior sample homogenization in the lysis buffer (data not shown).
Concerning the RNA extraction step, Lee et al have recommended to add a second phase separation and to use a high salt solution for precipitation. 14 We have found that a second phase (A) (B) FIGURE 4 Real-time polymerase chain reaction Ct values of endogenous control genes for the NP, inner AF, and outer AF tissues of bovine discs, according to the method used to isolate the RNA from each sample. Data are represented as mean AE SD; n = 3 to 6. Statistical differences are represented as described in Figure 2 separation is not required when a pronase predigestion step is performed prior RNA extraction and that the use of a high salt solution for RNA precipitation does not seem to have a major influence on RNA yield or purity when pronase predigestion is performed. If the phase separation is suboptimal, we recommend mixing the sample again, split it into 2 tubes, add 0.5 mL of TRIzol to each tube and continue with the usual RNA isolation procedure for TRIzol samples. At the end of the RNA extraction, the 2 tubes from the same samples are pooled (the pellet in the first tube is re-hydrated and the solution transferred to the second tube).
It is worth noting that only with the pronase predigestion step, the 260/230 ratio of RNA isolated from bovine NP was~1.5, while even with extra phase separation and use of high salt solution the 260/230 ratio was <0.8 (RIN not reported). 14 Therefore, we conclude that the pronase predigestion is a key step to obtain not only samples of high yield but also of high purity in all terms: RIN, 260/280 and 260/230 ratios. Pronase is a mixture of several nonspecific proteases that digest proteins down to single amino acids. Pronase is often used prior collagenase treatment for isolation of IVD cells. We hypothesize that pronase will cause partial extracellular matrix (ECM) loosening.
Real-time polymerase chain reaction dCt values of genes of interest for the NP, inner AF, and outer AF tissues of bovine discs, according to the method used to isolate the RNA from each sample. 18S was used as endogenous control; data are represented as mean AE SD; n = 3 to 6; n.d., gene not detected. Statistical differences are represented as described in Figure 2 Hence, some ECM will be removed already during the washes and centrifugation steps prior sample freezing. Moreover, pronase predigestion may contribute to a better exposure of the cells to the lysis buffer, which will in turn help to achieve a good phase separation and reduce the risk of coprecipitation of proteoglycans with RNA during the RNA extraction process.
In summary, we recommend: • 100 to 150 mg tissue per sample • predigestion in 0.2% pronase (5 mL/100 mg tissue) for 1 hour at 37 C on a shaker • pulverization of IVD tissues in liquid nitrogen • lysis in TRIzol (1 mL/100 mg of initial tissue weight) • homogenization (25 Hz, 6 min) • 1 phase separation In the predigestion and pulverization method, a short digestion (1 hour) is proposed, which will not affect the expression of most genes of interest. In the present study, some variations were also observed in the dCt values of certain genes ( Figure 5). These variations might be explained by the differences in the purity of the samples that may have influenced the amplification efficiency of certain primers to a variable extent. Nevertheless a possible effect of the enzyme treatment on the gene expression profile cannot be completely excluded. A previous study, comparing rat NP and AF marker genes between RNA extracted from isolated cells and RNA extracted directly from tissues, did not reveal any significant differences in the relative gene expression levels. 11 Further experiments would be required to investigate this subject on a whole transcriptome level.
As an alternative method, tissue pulverization in liquid nitrogen followed by RNA extraction in guanidinium thiocyanate-phenolchloroform and further purification of the aqueous phase with silica columns have been reported as a way to eliminate contaminants due to the coprecipitation of ECM components during RNA extraction. 11 However, in the present study this method led to lower yield and quality compared to predigestion and pulverization.
As for the gene expression, higher Ct values were observed for the endogenous control genes in the pulverization only group, which could be due to low yield and/or the possible presence of contaminants (eg, proteoglycans from ECM, TRIzol carry-over) which may affect both the reserve transcription and the RT-PCR. For instance, it has been shown that proteoglycans can act as inhibitors of PCRs. 15 In conclusion, this new combined method (predigestion and pulverization) improves the yield and quality of the RNA extracted from IVD tissues (NP, inner and outer AF) compared to pulverization alone. As a guideline when a RIN analyzer is not available (and based on the results displayed in Figure 3), the reference values for RNA evaluation with Nanodrop could be set as 20 ng/μL for RNA concentration and 260/280 ratio >1.8. This is achievable even for NP tissue using the predigestion and pulverization method. This method is of importance for gene expression studies on IVD degeneration and regeneration, including the most recent strategies such as rejuvenating the aging disc using stem cells and enhancing the endogenous repair of the disc by injecting an appropriate stimulating factor. Given the similarity in glycosaminoglycan 23 and collagen 24 content of IVD tissues across various species, we presume that the proposed RNA isolation method can be applied to IVD samples from different species. Moreover, the results obtained here using young bovine IVDs could be translatable to IVD tissues obtained from older animals. Indeed, with aging the collagen content is increasing at the expense of glycosaminoglycans. Since the latter are the strong contaminants in RNA purification, extraction of RNA from IVD of older animals may not be more challenging compared to younger animals. Nevertheless, direct comparison of all methods among animals of different age groups will be required to address this question. Finally, we also anticipate that this method may be further applied to other tissues rich in proteoglycans.
interpretation of data, drafting of manuscript and critical revisions.