Effect of duration of fixation with formalin on mRNA expression using quantitative RT-PCR

Abstract

Background:

Analysis of mRNA in archival tissues using RT-qPCR has the potential to become an important element in diagnosis and research. There is uncertainty whether mRNA extraction and analyses from archival tissues are possible or not. This study will look for the possibility of mRNA extraction, RT-qPCR analysis, and standardization of the protocol using formalin fixed paraffin-embedded blocks.

Objectives:

1. To determine the effect of 24-hour and 72-hour formalin-fixed paraffin-embedded tissue on the quantity and quality of mRNA. 2. To compare the quantity and quality of mRNA in fresh frozen tissues. 3. To compare the extracted mRNA expression using RT-qPCR in the above groups.

Methodology:

Twelve tissue samples were collected from patients undergoing minor surgical procedures and grossed into 3 bits. Each bit was placed in 24 hours of formalin and 72 hours of formalin, and the last bit was freezed in RNAlater at -80°C (positive controls), respectively. Routine tissue processing and sectioning was done followed by wax removal for the formalin-fixed tissues, and mRNA extraction using TRIzol method was done for all three groups. Extracted mRNA was quantified using Nanodrop spectrophotometer and its quality checked on mRNA TapeStation. cDNA synthesis was done followed by RT-qPCR analysis.

Results:

mRNA could be isolated with satisfactory quantity in all three groups. mRNA quality was significantly low for formalin-fixed tissues. But the RT-qPCR values of the two formalin groups were comparable to those obtained in fresh frozen tissues (P value = 0.00002).

Conclusion:

mRNA can be extracted from archives of paraffin tissue blocks that can be utilized to carry out enormous studies using RT-qPCR.

INTRODUCTION

Formalin fixation and paraffin embedding (FFPE) have become the gold standard for preserving tissue samples in diagnostic pathology.[1] This method allows long-term storage of tissue specimens, which is more cost-effective compared to storing tissues at ultra-low temperatures, representing a rich repository for researchers to explore disease mechanisms, biomarkers, and therapeutic targets.[2] Moreover, FFPE samples are often accompanied by extensive clinical data, such as patient outcomes and treatment histories, further enhancing their utility in research studies.[1,2]

Since antigen retrieval was first introduced to facilitate immunohistochemistry (IHC) on formalin-fixed paraffin-embedded samples, these FFPE blocks have been utilized for extracting DNA and proteins, which were then analyzed using Southern blotting, Western blotting, and real-time polymerase chain reaction (RT-PCR).[3] These methodologies have played a pivotal role in discovering new biomarkers that offer enhanced insights into prognosis and treatment options. Extracting mRNA from FFPE blocks and subjecting it to diverse assays hold great promise for furthering research endeavors. But mRNA extraction from these blocks and subjecting this mRNA to RT-PCR has not been achieved globally.[4]

The challenge of extracting mRNA from FFPE blocks stems from numerous pre-analytical variables, including warm and cold ischemia durations, fixative type, fixation duration, tissue sample size, transportation conditions, tissue processing methods, and, notably for biobanks, the storage conditions of the samples.[5] Currently, the pressing necessity lies in standardizing all the above variables. By doing so, we can effectively harness the vast repository of tissue samples available to us. Another difficulty with the use of mRNA from FFPE blocks is that the mRNA is too degraded for classical, quantitative analysis methods such as Northern blots. RT-PCR has been widely employed for gene expression detection in both cultured cells and fresh or frozen tissues.[4] Additionally, RT-PCR’s capability to assess minute mRNA fragments renders it suitable for investigations involving moderately or highly degraded mRNA, such as in FFPE tissue samples.[6]

The objective of this study is to develop a comprehensive protocol for the standardized processing of tissues, encompassing key steps such as making formalin-fixed paraffin-embedded (FFPE) blocks, mRNA extraction, and subsequent analysis via RT-qPCR. By establishing a robust and reproducible protocol, this research aims to provide a clear set of guidelines that can be universally adopted. This standardized approach will not only enhance the reliability and consistency of results but also facilitate comparability across different studies, ultimately advancing the utilization of tissue samples for molecular research and biomarker discovery.

METHODOLOGY

Ethical clearance was obtained from Institutional Ethical Clearance Committee with the number IEC-ACM (2)/10/2020-21. Patient’s consent was taken before collecting their tissue samples following minor surgical procedures. Twelve soft tissue samples measuring approximately 8 × 8 mm were collected and grossed into 3 bits and grouped as follows:

• Group I and II: 12 tissues fixed in formalin for 24 hours and 12 tissues fixed in formalin for 72 hours, respectively

• Group III: 12 tissues kept in RNAlater and freezed in -80°C (Control group)

Tissue processing

Following fixation for either 24 hours or 72 hours, tissues were transferred to processing cassettes and washed in running tap water for 30 minutes. They were then dehydrated in graded isopropyl alcohol (50%, 70%, 90%, and 100%) for 45 minutes each. Subsequently, tissues underwent clearing using graded xylene (50%, 90%, and 100%) for 30 minutes each. Once fully cleared, tissues were immersed in a wax bath and incubated overnight at 65°C in a hot air oven. The following day, tissues were embedded using an embedding machine (Leica EG1150 H). The resulting formalin-fixed paraffin-embedded (FFPE) blocks were stored until further use.

mRNA extraction

From the tissue blocks, 10-μm-thick sections were cut using a semi-automatic microtome and transferred to 1.5 ml Eppendorf tubes with a soft brush [Figure 1]. The tubes were placed at 65°C in an incubator for 5 minutes, followed by addition of 1 ml of xylene and another 5-minute incubation at 65°C. This xylene treatment was repeated 2-3 times to remove wax completely. The tissue sections were then rehydrated using graded ethanol (100%, 90%, 70%, 50%, and 30% ethanol), with each step lasting 2 minutes, and then briefly washed with 1 ml 1xPBS. Concurrently, positive controls preserved in mRNAlater were included in the study after removing the mRNAlater. Next, 500 μl TRIzol was added to all the tissue bits and stored at -80°C overnight. Thawed samples were homogenized at 4-8 RPM at 4°C, followed by addition of 100 μl chloroform and vigorous vortexing to create two layers. Centrifugation at 14,000 rpm for 15 minutes at 4°C was done, and the top aqueous layer containing mRNA was transferred to new tubes [Figure 2]. To precipitate mRNA, an equal volume of isopropanol was added and mixed gently, followed by addition of 1 μl glycogen for pellet visibility [Figure 3]. The tubes were centrifuged at 15,000 rpm for 30 minutes at 4°C, and the supernatant was carefully removed. The mRNA pellet was washed twice with 500 μl 80% ethanol in diethyl pyrocarbonate (DEPC) water, air-dried for 5 minutes, and resuspended in 20 μl DEPC water. Finally, the mRNA was dried at 65°C for 3 minutes on a heating block and was ready for quantification, quality assessment, and cDNA synthesis. It is crucial to store mRNA at -80°C until further use to maintain its integrity.

Figure 1.

Tissue sections collected in Eppendorf tubes

Figure 2.

Layers formed after centrifuging

Figure 3.

RNA pellet formed at the bottom

mRNA quantification using Nanodrop spectrophotometer

1 μl of DEPC water was first put in the well of Nanodrop quantifier, and a 0 reading was obtained (blanking achieved). 1 μl from each sample was put on the well one after the other carefully avoiding contamination. The readings for each sample were noted down [Figure 4].

Figure 4.

Nanodrop spectrophotometer showing the RNA concentration

RNA quality analysis using the Agilent TapeStation

RNA analysis using the Agilent TapeStation began by equilibrating mRNA Sample Buffer at room temperature for 30 minutes. RNA ladder and samples were thawed on ice. The Agilent TapeStation Controller software was launched, and the mRNA ScreenTape device was inserted into the instrument. Sample positions were selected, and consumables were displayed in the software. Reagents and samples were vortexed and spun down, and then, 5 μL of mRNA sample buffer and 1 μL of mRNA ladder were pipetted into position A1. Each tube strip received 5 μL of mRNA sample buffer and 1 μL of mRNA sample, capped, and centrifuged at 2000 rpm for 1 minute. Samples were heated at 72°C for 3 minutes, cooled on ice for 2 minutes, and then centrifuged for 1 minute. After loading into the TapeStation, the run started with the ladder in position A1. Caps were removed, ensuring liquid at the tube bottoms. RNA integrity number (RIN) values were recorded [Figure 5].

Figure 5.

RNA run on TapeStation

cDNA synthesis from mRNA

Based on the average mRNA quantity observed using Nanodrop quantifier, 5 μg mRNA was taken for cDNA conversion from each sample. The calculations were done accordingly:

1 microgram = 1000 nanogram, 5 micrograms = 5000 nanogram

For e.g., the calculation for one sample is as follows,

503.9 nanograms in 1 microliter, 5000 nanogram in

Seeing the maximum volume from all samples, the total volume to be taken was decided as 20μl. To make the total volume of 20μl, nuclease-free water (NFW) was added to each sample. 0.5μl DNase1 enzyme and 0.5μl DNases buffer were added to each tube.

Total volume of (20μl + 0.5μl + 0.5μl) 21μl of each sample was then incubated at 37°C for 10 mins and at 75°C for 10 mins inside the PCR (thermocycler) machine.

Add 1μl random hexamer and 1μl dNTP to the tubes. The total volume of (21μl + 1μl + 1μl) 23μl of each sample was then incubated inside thermocycler at 65°C for 5 mins and immediately kept at 4°C. 4μl 5xprime buffer, 0.5μl mRNase1, and 1.0μl RTase and additional nuclease-free water were added to get 50μl of cDNA in each tube. Immediately, the tubes containing cDNA were transferred to -20°C.

Primer selection

GAPDH is usually taken as the housekeeping gene in RT-qPCR studies, and β-catenin was chosen as it is commonly observed in mRNA studies. Primer selection and standardization were done using NCBI primer blast website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and base pair size was kept below 200 bp. The specific primers sequences of both forward (F) and reverse (R) were commercially obtained from JUNIPER Life Sciences Company in a lyophilized form for GAPDH and β-catenin.

Preparation of primer stock and working solution

As per the manufacturer’s instruction, molecular grade water was added to both the forward and reverse primers to make 100 μM stock solution. 10 μl of forward primer stock and 10 μl of reverse primer stock were added to 80 μl molecular grade water to make 10 μM of working solution.

Preparation of the master mix for RT-qPCR setup

A total volume of 10 μl master mix was needed in each well of the RT-PCR plate. Hence, the following values of each reagent were chosen: DEPC water is added to make up the solution up to 10 microliters

cDNA (obtained from the tissue samples): 0.5 microliter

Primer : 1 microliter

ROX (in kit) : 0.2 microliter

SYBR green (ELK Technologies) : 5 microliter

DEPC water : 3.3 microliter.

RT-qPCR setup

Trial runs were done to standardize the amount of cDNA needed and to check if the primers are working. Each sample was taken in triplicate. GAPDH gene was taken as housekeeping gene. Initially, 1 μl of cDNA was taken. The amplification curve began below the threshold value. Hence, the cDNA was diluted further to 1:5 and 1:10 with DEPC water. 1:10 dilution with 1 μl of cDNA and 1 μl primer gave good results and was standardized for further RT-PCR runs.

Running real-time polymerase chain reaction machine

The qPCR reaction components were added to 96-well plate in triplicate and then placed inside the qPCR machine for amplification. Primer annealing temperature was set at 60°C. Each amplification reaction cycle consisted of 95°C for 5 minutes for initial denaturation. Extension at 58°C for 30 seconds and final extension at 95°C for 5 mins followed by cooling for 5 mins. Samples were amplified for 40 consecutive cycles. In every well, the qPCR experiment measured the expression intensity of the particular genes as cycle threshold (Ct) values in amplification curve [Figure 6].

Figure 6.

Amplification curve obtained in RT-qPCR

Statistical analysis

The mean of all parameters between the three groups was compared using one-way ANOVA test at p value < 0.05. Pairwise comparison between two of the three groups was done using post hoc Tukey HSD beta test with a significant p value at < 0.05.

RESULTS

The mRNA was successfully extracted from all 36 samples, including positive controls. The yield from FFPE blocks was significantly lower than that from fresh tissue [p = 0.000238, one-way ANOVA; Table 1a], as quantified using a Nanodrop spectrophotometer. However, no significant difference was observed in the A260/280 purity ratios among the three groups. Post hoc Tukey HSD analysis revealed no significant differences in mRNA quantity or purity between 24-hour and 72-hour formalin fixation groups [p = 0.9997; Table 1b].

Table 1a.

RNA concentration obtained in Nanodrop spectrophotometer

Groups	No. of samples	Mean of RNA conc. (ng/µl)	Mean of A260/280	Standard deviation	F	P (one-way ANOVA)
Group I	12	77.5583	1.8	137.8049	F=10.856	0.000238
Group II	12	79.2308	1.7	125.874
Group III	12	370.075	1.9	243.3233

Table 1b.

Pairwise comparison of RNA concentration using post hoc Tukey HSD (beta)

Pairwise comparison	HSD.05=177.3622 HSD.01=226.0446	Q.05=3.4702 Q.01=4.4227
G1=77.56 G2=79.23	1.67	Q=0.03 (P=0.99970)
G1=77.56 G3=370.07	292.52	Q=5.72 (P=0.00084)
G2=79.23 G3=370.07	290.84	Q=5.69 (P=0.00090)

RNA integrity numbers (RIN) assessed by RNA TapeStation were markedly lower in FFPE samples compared to fresh frozen tissues, indicating mRNA degradation [p < 0.0001; Table 2a], with no significant difference between the 24-hour and 72-hour fixation groups [p = 0.671; Table 2b]. Despite reduced integrity, mRNA quantity and quality were adequate for RT-qPCR analysis.

Table 2a.

RIN value obtained using RNA TapeStation

Groups	No. of samples	Mean of RIN value	Standard deviation	F	P (one-way ANOVA)
Group I	12	2.8417	0.3204	F=41.25	<0.0001
Group II	12	3.0089	0.2875
Group III	12	4.45	0.7052

Table 2b.

Pairwise comparison of RIN value using post hoc Tukey HSD (beta)

Pairwise comparison	HSD.05=177.3622 HSD.01=226.0446	Q.05=3.4702 Q.01=4.4227
G1=2.84 G2=3.01	0.17	Q=1.21 (P=0.671)
G1=2.84 G3=4.45	1.61	Q=11.68 (P=0.000)
G2=3.01 G3=4.45	1.44	Q=10.47(P=0.000)

Ct values showed no significant differences across the three groups [p = 0.765; Table 3a] or between the 24-hour and 72-hour fixation subgroups [p = 0.989; Table 3b], indicating that mRNA extracted from both fixation durations is suitable for RT-qPCR applications.

Table 3a.

Ct values of β-catenin in RT-qPCR

Groups	No. of samples	Mean of Ct values	Standard deviation	F	P (one-way ANOVA)
Group I	12	17.8571	4.3765	0.269	0.765
Group II	12	18.0433	3.2258
Group III	12	18.7733	1.3352

Table 3b.

Pairwise comparison of Ct values using post hoc Tukey HSD (beta)

Pairwise comparison	HSD.05=3.2379 HSD.01=4.1267	Q.05=3.4702 Q.01=4.4227
G1=17.86 G2=18.04	0.19	Q=0.20 (P=0.989)
G1=17.86 G3=18.77	0.92	Q=0.98 (P=0.768)
G2=18.04 G3=18.77	0.73	Q=0.78(P=0.845)

DISCUSSION

mRNA is highly susceptible to degradation due to the omnipresence of Rnases, necessitating stringent precautions in laboratory settings to avoid contamination. These enzymes are pervasive, even on human skin, highlighting the need for gloves and RNase-free equipment during RNA extraction. Purified mRNA remains vulnerable and must be handled delicately to prevent rapid breakdown.[7] For tissue samples, immediate immersion in fixatives like 10% formalin or RNAlater is crucial to preserve RNA integrity, mitigating potential changes in gene expression caused by surgical trauma, hypoxia, or fixation-related stress. Proper handling, including sterilization of glassware at 150°C and treatment of plasticware with sodium hypochlorite followed by autoclaving, is essential to maintain mRNA quality throughout experimental procedures.[8] Even if mRNA as molecules is preserved, it is possible that gene expression is changed during transport of non-fixed tissues. Other factors that alter gene expression are surgical trauma, hypoxia, and the effects of fixation chemicals diffusing into tissues.[9] To mitigate mRNA loss before fixation, tissues in this study were promptly immersed in fixatives like 10% formalin and RNAlater. The 10% formalin used was prepared by diluting commercially available formaldehyde solution in a 1:10 ratio with tap water.

Control samples were preserved in RNAlater, a dependable solution known for stabilizing mRNA and preserving expression profiles. It is compatible with various RNA isolation methods such as TRIzol Reagent, GenElute Total mRNA isolation kits, and mammalian mRNA isolation kits.[10] RNAlater can maintain RNA integrity for up to 1 day at 37°C, 1 week at 25°C, and 1 month at 4°C, allowing tissues to be stored long term at -20°C.[8]

In this study, tissues were stored at -80°C for several months prior to processing. Fresh frozen tissue is usually the preferred sample to detect gene mutation due to its superiority in preserving mRNA. However, fresh frozen tissue is often not available in clinical practice, as the associated protocol requires that resected tissue be snap-frozen in liquid nitrogen 30–60 min after surgical resection and due to many other reasons like cost, storage, maintenance, etc., Compared with fresh frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue has several advantages: (1) preservation of the cellular and architectural morphology; (2) the possibility of storage at room temperature for several years; and (3) easy availability, as FFPE blocks are routinely prepared in the pathology departments of most centers.[11] In accordance with several such studies, this study also utilized FFPE blocks that were customized according to the fixation time of 24 hours and 72 hours to check for mRNA quantity and quality.

The Nanodrop spectrophotometer measures how much UV light passes through the sample after absorption at 260 nm. Higher absorbance at 260 nm indicates a higher concentration of nucleic acids in the sample. A ratio of A260/280 is used to estimate the amount of protein contamination in the nucleic acid sample. For pure RNA, the ideal A260/280 ratio is typically between 1.8 and 2.0.[12] Our formalin-fixed samples showed mRNA concentration lower than that of fresh frozen tissues but sufficient enough for RT-qPCR analysis. The A260/280 ratio of purity was in range with fresh samples taken in the study. It indicates that the quantity of mRNA may be reduced but purity can still be maintained in FFPE blocks.

The RNA integrity number (RIN) is a measure used to assess the quality of mRNA samples. The RIN score ranges from 1 to 10, with 1 being the lowest quality and 10 being the highest.[13] mRNA with low RIN scores (like 2 to 3) may still be used in certain studies but requires careful consideration and validation to ensure results are meaningful and not biased by mRNA degradation effects. The mRNA quality was considerably low for formalin-fixed tissues as indicated by RIN in the range of 2 to 3 which is toward the lower range and quality. mRNA with low RIN scores like 2 to 3 may still be used in studies but requires careful consideration and validation to ensure results are meaningful and not biased by mRNA degradation effects. But this degraded mRNA can be used in RT-qPCR studies with appropriate dilutions as mentioned in this study, provided the gene of interest has an amplicon size of less than 200 base pairs. The cycle threshold (Ct) values have come below the threshold value of fresh frozen tissues which indicates early detection of mRNA due to its abundance in the sample. On further dilution of the sample, the Ct value may rise in accordance with the fresh samples.

CONCLUSION

The present study elaborates that tissues fixed in formalin can be a source of mRNA for research studies or for diagnostic purposes in clinical workflow provided RT-qPCR is used to check for mRNA expression. Even though the mRNA is considerably degraded, it is not completely lost during the process of fixation and tissue processing. Duration of fixation and paraffin embedding does not hamper the mRNA extraction given that the procedure adopted and standardized in this study be followed. Further studies involving FFPE blocks stored for different months and years can be compared and studied.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.