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. 2010 Jan;15(1):80-5.
doi: 10.1177/1087057109355059. Epub 2009 Dec 11.

Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis

Sean T Glenn  1 , Karen L Head, Bin T Teh, Kenneth W Gross, Hyung L Kim
Affiliations

Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis

Sean T Glenn et al. J Biomol Screen. 2010 Jan.

Abstract

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen's TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers' protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan qPCR can be optimized by using the MasterPure RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

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Figures

Figure 1
Figure 1. Determination of optimal RNA isolation method for use with archival renal tissue
(A) Comparison of RNA yields from archival renal tumors using 5 different methods for RNA isolation, including 4 commercial kits. Using each method, RNA was isolated from three 10 µm sections from 10 FFPE renal tumors. The total RNA recovered was diluted to a constant volume before measuring RNA concentration using a flourometric method. The Epicentre kit had the highest yield of RNA and the Roche kit had the lowest yield. Paired t-test was performed comparing the indicated groups. (B) Comparison of standard vs. overnight Proteinase K digestion. The 3 top-performing kits were used either with a standard 3 hour Proteinase K digestion or an overnight digestion. The overnight digestion significantly increased RNA yield from the FFPE tissues for all 3 kits. Error bars are not provided since the variability in RNA yield between tumors is greater than the variability in RNA resulting from use of different isolation methods. Paired t-test was performed comparing the 2 Proteinase K digestion groups.
Figure 1
Figure 1. Determination of optimal RNA isolation method for use with archival renal tissue
(A) Comparison of RNA yields from archival renal tumors using 5 different methods for RNA isolation, including 4 commercial kits. Using each method, RNA was isolated from three 10 µm sections from 10 FFPE renal tumors. The total RNA recovered was diluted to a constant volume before measuring RNA concentration using a flourometric method. The Epicentre kit had the highest yield of RNA and the Roche kit had the lowest yield. Paired t-test was performed comparing the indicated groups. (B) Comparison of standard vs. overnight Proteinase K digestion. The 3 top-performing kits were used either with a standard 3 hour Proteinase K digestion or an overnight digestion. The overnight digestion significantly increased RNA yield from the FFPE tissues for all 3 kits. Error bars are not provided since the variability in RNA yield between tumors is greater than the variability in RNA resulting from use of different isolation methods. Paired t-test was performed comparing the 2 Proteinase K digestion groups.
Figure 2
Figure 2. Comparison of methods for RNA quantification
(A) Serial dilutions of total RNA from 3 tumor samples were quantified using the Nanodrop ND-1000. RNA concentrations measured using the Nanodrop were compared to qPCR Ct for ACTB, which was considered the “gold standard”. The log linear plot of Ct vs. concentration showed excellent correlation with R2 of 0.9273 for the log fit. Therefore, the Nanodrop accurately reflects RNA concentration. (B) The same serial dilutions and samples were quantified using the RediPlate 96 RiboGreen RNA quantification kit. The concentrations measured by the RediPlate kit were then compared to the Ct values for ACTB. The log linear plot also showed excellent correlation with R2 of 0.9047. Therefore, the RediPlate accurately reflects RNA concentrations.
Figure 3
Figure 3. Strategies to increase the sensitivity of qPCR
(A) Scatter plot of qPCR data following reverse transcription reactions using random or specific primers (Table 1). Reverse transcription was carried out with RNA from 6 FFPE renal tumors using either random or gene-specific primers. The average threshold cycle from the qPCR reactions for each of the 23 genes analyzed was plotted to compare the 2 different methods for reverse transcription. Use of specific primers in the RT reaction increased the level of cDNA measured using qPCR. The R2 was 0.9181, indicating a strong correlation. Therefore the use of specific primers maintains the ability of qPCR to quantify gene expressions. (B) Comparison of TaqMan® Custom Arrays (TCA) and 384-well 5ul qPCR reactions set up using a robotic liquid handler. The Ct for 63 genes were plotted for both the TCA and the standard qPCR reactions. The primers/probes used in the conventional qPCR were designed in-house using Bio-Rads Beacon Designer 3 software. TCA cards were custom-built by Applied Biosystems to assay the same 63 genes. For the majority of genes assessed, conventional qPCR detected transcripts at lower cycles, indicated higher sensitivity. The error bars indicate standard error of the mean for experiments performed in triplicate. (C) Comparison of Bio-Rad CFX384 and ABI 7900HT qPCR systems. Identical 384-well plates were created and qPCR reactions were performed using the 2 qPCR systems. The ABI 7900HT system detected the threshold cycle values earlier, indicating greater sensitivity for detecting transcripts when compared to the Bio-Rad system. The error bars indicate standard error of the mean for experiments performed in triplicate.
Figure 3
Figure 3. Strategies to increase the sensitivity of qPCR
(A) Scatter plot of qPCR data following reverse transcription reactions using random or specific primers (Table 1). Reverse transcription was carried out with RNA from 6 FFPE renal tumors using either random or gene-specific primers. The average threshold cycle from the qPCR reactions for each of the 23 genes analyzed was plotted to compare the 2 different methods for reverse transcription. Use of specific primers in the RT reaction increased the level of cDNA measured using qPCR. The R2 was 0.9181, indicating a strong correlation. Therefore the use of specific primers maintains the ability of qPCR to quantify gene expressions. (B) Comparison of TaqMan® Custom Arrays (TCA) and 384-well 5ul qPCR reactions set up using a robotic liquid handler. The Ct for 63 genes were plotted for both the TCA and the standard qPCR reactions. The primers/probes used in the conventional qPCR were designed in-house using Bio-Rads Beacon Designer 3 software. TCA cards were custom-built by Applied Biosystems to assay the same 63 genes. For the majority of genes assessed, conventional qPCR detected transcripts at lower cycles, indicated higher sensitivity. The error bars indicate standard error of the mean for experiments performed in triplicate. (C) Comparison of Bio-Rad CFX384 and ABI 7900HT qPCR systems. Identical 384-well plates were created and qPCR reactions were performed using the 2 qPCR systems. The ABI 7900HT system detected the threshold cycle values earlier, indicating greater sensitivity for detecting transcripts when compared to the Bio-Rad system. The error bars indicate standard error of the mean for experiments performed in triplicate.
Figure 3
Figure 3. Strategies to increase the sensitivity of qPCR
(A) Scatter plot of qPCR data following reverse transcription reactions using random or specific primers (Table 1). Reverse transcription was carried out with RNA from 6 FFPE renal tumors using either random or gene-specific primers. The average threshold cycle from the qPCR reactions for each of the 23 genes analyzed was plotted to compare the 2 different methods for reverse transcription. Use of specific primers in the RT reaction increased the level of cDNA measured using qPCR. The R2 was 0.9181, indicating a strong correlation. Therefore the use of specific primers maintains the ability of qPCR to quantify gene expressions. (B) Comparison of TaqMan® Custom Arrays (TCA) and 384-well 5ul qPCR reactions set up using a robotic liquid handler. The Ct for 63 genes were plotted for both the TCA and the standard qPCR reactions. The primers/probes used in the conventional qPCR were designed in-house using Bio-Rads Beacon Designer 3 software. TCA cards were custom-built by Applied Biosystems to assay the same 63 genes. For the majority of genes assessed, conventional qPCR detected transcripts at lower cycles, indicated higher sensitivity. The error bars indicate standard error of the mean for experiments performed in triplicate. (C) Comparison of Bio-Rad CFX384 and ABI 7900HT qPCR systems. Identical 384-well plates were created and qPCR reactions were performed using the 2 qPCR systems. The ABI 7900HT system detected the threshold cycle values earlier, indicating greater sensitivity for detecting transcripts when compared to the Bio-Rad system. The error bars indicate standard error of the mean for experiments performed in triplicate.
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