An optimized protocol for total RNA isolation from archived formalin-fixed paraffin-embedded tissues to identify the long non-coding RNA in oral squamous cell carcinomas

Highlights • TRI reagent modified protocol for RNA isolation is cost-effective compared to the kit method.• The Quality and quantity of isolated RNA is better in the TRI reagent modified protocol compared to the kit method.• The TRI reagent modified procedure has fewer steps than the kit method without requiring any additional training or time.


Specifications
( continued on next page )

Introduction
The long non-coding RNAs (lncRNAs) are a class of RNAs > 200 nucleotides length. These lncRNAs are emerging as novel players in the field of cancer diagnostics or prognostics as they are involved in oncogenic and tumor-suppressive regulatory functions [1] . Recently, lncRNAs dysregulation has been associated with oral squamous cell carcinomas (OSCC) and has been known to affect various aspects such as cellular homeostasis, proliferation, survival, migration, or genomic stability [2] . However, there is scarcity of literature reports on association of dysregulated lncRNAs with head and neck squamous cell carcinoma (HNSCC).The functional significance of lncRNAs specifically in OSCC has been remained unexplored [3] .
The ability to predictably retrieve sufficient RNA for cDNA template generation and subsequent quantitative polymerase chain reaction (qPCR) facilitates differential gene transcriptional analysis [4] . Recent introduction of high-content, high-throughput Quantitative Real time PCR (qRT.-PCR) has demonstrated that RNA extracted from FFPE tissue sections could produce reliable qRT-PCR data [ 5 , 6 ]. Short RNA fragments like miRNA are stable and detectable in qRT-PCR in FFPE tissues. Literature reports have revealed the reliable expression levels of miRNA in FFPE as compared to paired fresh-frozen samples [ 7 , 8 ]. However, survivability and expression level of lncRNA in FFPE tissues as compared to fresh tissues is not well documented in the literature. This could be owing to the longer length, degradation, and fragmentation of lncRNA associated with fixation and processing [5] .
With this background, we designed the current pilot study with the main aim to optimize the modified TRI reagent RNA isolation protocol to identify few important lncRNA expression in archived FFPE tissues of OSCC. Normal mucosa was used as control. In addition, we also aimed to compare commercially available column-based RNA isolation kit viz. NucleoSpin, Total RNA FFPE, Germany with modified TRI reagent RNA isolation protocol to check for the quality, and its usefulness in lncRNA expression analysis.

RNA isolation procedure from FFPE by optimized TRI reagent modified protocol 1. FFPE tissue sectioning
• Take FFPE tissues sections of 6-8 μ thickness using soft tissue microtome.
• Clean the blades with xylene after each sample to remove paraffin residues. • Transfer 4 to 5 cut paraffin sections into 1.5 mL Eppendorf tubes.
Note: Slightly rolled up sections can be better handled and this could be obtained by decreasing the temperature of the paraffin blocks by placing ice on the cutting surface or putting paraffin blocks in a freezer before cutting.

Deparaffinization
• Add 1 ml of xylene, vortex, and then incubate in the water bath at 56 ˚C for 10 min. Note: To remove the paraffin and unmask hidden or latent epitopes in preparation for downstream application. The procedure of Xylene and absolute alcohol can be repeated to ensure the complete removal of paraffin from tissue.

Protein digestion
• Add proteinase K digestion buffer containing390 μl lysis buffer and 10 μl of proteinase K (500 μg/ml) and vortex it. • Incubate the tube in the water bath at 56 ˚C for 60 min, after incubation immediately transfer the tube into the ice. • Add 1ml TRIZOL reagent, vortex for 2-5 s, and incubate for 1-2 min at room temperature • Add 0.2 ml of chloroform, vortex, and incubate at room temperature for 5 min • Centrifuge at 13,0 0 0 rpm for 15 min at 4 °C and collect the supernatant.
Note: To digest proteins and remove contamination from nucleic acid preparations. Buffer was added in nucleic acid preparations for the inactivation of nucleases that could degrade RNA during isolation and purification applications. Carefully Removing the aqueous phase (supernatant) is a very crucial step and avoids contamination with the interphase and organic phase.  To the aqueous phase, Add 0.6 ml of isopropanol and incubate at -20 0 C for overnight for RNA precipitation.

RNA wash, solubilization and Pellet Drying
RNA pellet is washed with 100% ethanol, briefly air-dried. Pallet is dissolved in RNase-free water RNA pellet is washed in 75% chilled ethanol, dried in thermo mixer at 37 °C for 5 min. Pellet is dissolved in nuclease-free water Note: Isopropanol precipitation is based on the principle of salting out, in the presence of salts that renders nucleic acid preferentially to become insoluble and the precipitate is collected by centrifugation. The process also purifies the RNA leaving out alcohol soluble salts, organic solvents, and detergents. The addition of glycogen was not done in the present protocol as it may cause contamination. Maintaining temperature during incubation and optimal centrifugal force is important to avoid degradation of RNA.

Pellet drying
• Dry the pellet in thermomixer at 37 °C for 5min • The RNA was eluted in 30-50 μL of nuclease-free water Note: Ethanol should completely evaporate or else it prevents the RNA solubilization into nucleasefree water.
If the pellet dries out too much, the RNA crystallizes and is very difficult to resolubilize. Main modifications made in the present optimized protocol compared to TRI reagent baseline protocol [ 9 ] are mentioned in Table 1 .

RNA isolation procedure from FFPE by the kit method
RNA isolation was also carried out from the study samples using a column-based kit (NucleoSpin, Total RNA FFPE, Germany) following the manufacturer's protocol.

Estimation of RNA concentration and quality
The concentration of RNA was estimated at 260:280 absorbance using a Bio-Spectrophotometer (Eppendorf, model no.6136, Germany) in nanograms. Then the RNA was stored in aliquots of the required quantity at -20 °C in Eppendorf tubes sealed with parafilm.
After the RNA quantification, the integrity of RNA was verified using 200 ng of RNA in 1% formaldehyde agarose gel electrophoresis in 1X MOPS buffer at 100 V for 30 min and stained with Table 2 The primers sequence of lncRNAs and endogenous control gene.

HOTAIR-F
GAPDH-F GGGGAAGGTGAAGGTCGGAG GAPDH-R ACGGTGCCATGGAATTTGCC ethidium bromide (ETBR) to visualize the RNA bands. This helps to know the quality of the extracted RNA and also to determine the presence of any contaminants like DNA or Protein.

cDNA synthesis
All RNA extracts were prepared at 1μg/μl per sample and transcribed into cDNA using Prime script 1 st strand cDNA synthesis kit (Takara, Japan) as per the manufacturer's instructions. DNA sample was diluted with nuclease-free water (1:10) and stored at -20 °C until further use.

Quantitative real time PCR (qRT-PCR)
qRT-PCR (Rotor Gene Q MDx) was performed with TB green Mix (Takara, Japan) in a total volume of 20 μl. Primer sequence for lncRNAs and endogenous control gene (GAPDH) used to normalize the expression level were custom-designed ( Table 2 ). Primers were designed using software Tool ( https://bioinfo.ut.ee/primer3-0.4.0/ ) and were checked for specificity using basic local alignment search tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ).The ct values of study cases were compared in TRI reagent modified protocol and kit method. Agarose gel electrophoresis was carried out to determine the molecular weight of the qRT-PCR end product .
The flow chart of the work carried out is represented in Fig. 1 .

Comparison of total RNA isolation methods (quantity & quality)
The total RNA concentrations using TRI reagent modified protocol was better than the kit method ( Table 3 ). The A260:A280 ratio was between 1.71 to 1.92 indicated the good quality RNA in both RNA isolation methods ( Table 3 , Fig. 2 ). The integrity of RNA was verified by resolving in 1% formaldehyde agarose gel electrophoresis. Bands were visualized by staining in ethidium bromide stain (HiMedia, Mumbai). ( Fig. 3 ).

qRT-PCR analysis and melting curve analysis
The cyclic threshold values (ct values) of study cases in TRI reagent modified protocol and kit method were found to be comparable and the mean difference between them was < 1 ( Figs. 4 and 5 ). Melting curve analysis was carried out to assess the specificity of each primer pair ( Fig. 6 ). Agarose gel electrophoresis of qRT-PCR product was carried out to confirm the molecular weight of lncRNAs which were < 200 nucleotide base pair units ( Figs. 7 and 8 ).

Conclusion
The present study results demonstrated that RNA quantity and quality was comparatively better with TRI reagent modified protocol than the kit method. The ct values after reverse-transcription and  qRT-PCR were comparable and almost equal in both the methods for normal mucosa (control) and OSCC samples.
Furthermore, the TRI reagent modified RNA isolation approach was cost-effective, costing roughly 1/3 of the cost of the kit method, and is particularly advantageous when there are a large number of samples. Additionally, while the column-based kit method took less time to isolate RNA, it required more steps, which could lead to human error.
The TRI optimized protocol for RNA isolation effectively demonstrated lncRNA expression in oral tissues without demanding any additional training or extra time and also saves money. Hence, we recommend to use TRI optimized method for RNA isolation in lncRNA expression studies using FFPE tissues.

Formaldehyde Agarose Gel Mix
Agarose -1 gm 10x MOPS buffer -10 ml Distilled Water -72 ml Note: Melt agarose in the oven, then let it to cool at 55 °C, then add 18 ml of Formaldehyde (37%). 1X Gel MOPS running Buffer 10 X MOPS buffer -100 ml 37% Formaldehyde -20 ml Distilled water -880 ml Total volume -10 0 0 ml Note: Make up a fresh buffer for each gel

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.