Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials - PubMed (original) (raw)

Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials

Jinghuan Li et al. BMC Biotechnol. 2008.

Abstract

Background: Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62-164 bp).

Results: Results from the Bioanalyzer and TaqMan data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70 degrees C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification.

Conclusion: Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.

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Figures

Figure 1

A schematic representation of the experimental procedures. Normal thyroid cell lines were split into two aliquots: One was snap frozen, the other formalin fixed and paraffin embedded. RNA was extracted using selected protocols and was then reverse transcribed into cDNA followed by different TaqMan® QRT-PCR analysis.

Figure 2

Qualities of RNA were measured using Agilent 2100 Bioanalyzer. (Str = Stratagene protocol; Am = Ambion protocol; + = modified with incubation in Proteinase K buffer at 70°C for 20 minutes; and SN = snap frozen.) Modification to the Stratagene and Ambion protocols generated larger fragments (lane 2 and 5) compared to the extracts generated using the original protocols (lane 1 and 4). Each well contained 1 μl of extracted RNA from equal amount of starting materials of 105 cells.

Figure 3

TaqMan® gene expression pattern using UPMM, GEMM and PreAmp. (Str = Stratagene protocol; Am = Ambion protocol; + = modified protocols with incubation in Proteinase K buffer at 70°C for 20 minutes; and SN = snap frozen cells. Not all assays produced products, e.g. HLA_A in UPMM.) CTs were higher using FFPE extracts compared to snap frozen counterparts when the amount of input RNA was identical. Modification to Stratagene and Ambion protocols produced decreased CTs. TaqMan® with UPMM generated higher CTs than GEMM and PreAmp when using longer amplicon lengths. This was particularly evident when comparing two GAPDH assays – one 122 bp and the other 67 bp. A mean difference of 17 cycles between small and large assays using UPMM and a mean difference of 4 cycles between small and large assays using GEMM was observed.

Figure 4

The effect of different incubation conditions on RNA. Total RNA was extracted from a large number (2 × 106 cells) of formalin fixed paraffin embedded cells using Ambion RecoverAll™ kit. Five eluted RNA aliquots from one extraction were subjected to different incubation conditions on a hot rack (lanes 6 to 10). In parallel, five eluted RNA aliquots from one snap frozen extraction were incubated (lane 1 to 5). Each well contained 1 μl of 4 ng/μl RNA in Ambion Elution Solution. Incubation conditions were as follows: 0 = no treatment; 70-10 = 70°C for 10 min; 70-20 = 70°C for 20 min; 95-10 = 95°C for 10 min and 95-20 = 95°C for 20 min. RNA from snap frozen preparations was not degraded at 70°C for 20 min (lane 3), but was degraded at 95°C (lane 4 and 5). RNA from FFPE showed large fragments and cross-linked RNAs which are approximately 5 kb (lane 6 – arrow). It was found that incubation of eluted RNA at 70°C for 20 min is the optimal condition, among these tested, to break up cross-links while not compromising RNA integrity.

Figure 5

Comparison of TaqMan® gene expression pattern using ΔCT method. (Am = Ambion protocol; FP = FFPE cells; + = modified protocols with incubation in Proteinase K buffer at 70°C for 20 minutes; and SN = snap frozen cells. Not all assays produced products, e.g. HLA_A in UPMM.) These ΔCT data was generated from the CTs shown in Figure 3. A Theoretical ΔCT was calculated for each chart based on equilibrating the results for any variation in input cDNA. In panel a, ΔCT = CT_UPMM - CT_GEMM. The Theoretical ΔCT of UPMM-GEMM was 0 [= Log2(20 ng/20 ng)] given identical input quantities (20 ng) were used in each system. In panel b, ΔCT = CT_UPMM - CT_PreAmp. In panel c, ΔCT = CT_GEMM - CT_PreAmp. The Theoretical ΔCT in panel b and c was 5.68 [= Log2(1024 ng/20 ng)], which was calculated based on an input of 1024 ng of cDNA for the TaqMan® real time PCR component of pre-amplification process. This quantity was generated from an initial 1 ng subjected to 10 cycles of pre-amplification with a 100% efficiency and no bias introduced from the PreAmp. The relevant Theoretical ΔCT is plotted on each chart as a reference point for measuring the actual detected ΔCT against the theoretically optimal ΔCT. The benefit of GEMM over UPMM was evident as amplicon size increased (Panel a). A comparison of UPMM and PreAmp showed a similar pattern (Panel b). However, PreAmp results generally correlated with GEMM regardless of amplicon size for the series of 8 assays analysed (Panel c).

Figure 6

Comparison of TaqMan® real time PCR with or without PreAmp using CDKN1B assay on extracts produced by different extraction protocols. (1 and 2 = Stratagene extracts; 3 and 4 = Ambion extracts; 5 and 6 = Gentra extracts; 7 and 8 = Trizol extracts; * = No amplification was achieved from FFPE) ΔCT = CT (no_PreAmp) - CT (with_PreAmp). The Theoretical ΔCT was 5.68 [= Log2(1024 ng/20 ng)] which was calculated based on an input of 1024 ng of cDNA for the TaqMan® real time PCR component of pre-amplification process and 20 ng of cDNA for the TaqMan® without pre-amplification. Ideally, ΔCT is in a range between Theoretical ΔCT +/- 1 which was achieved in this experiment indicating there is no bias in the PreAmp.

Figure 7

Analysis of GAPDH using two sizes of amplicon showing the difference of CTs between FFPE and snap frozen counterparts. ΔCT = CT (FFPE) - CT (Snap-Frozen). The smaller amplicon size of GAPDH (67 bp) generated lower ΔCTs than the larger amplicon size of 122 bp indicating that smaller amplicon sizes produced more comparable CTs in snap frozen and FFPE samples.

References

1. Lewis F, Maughan NJ, Smith V, Hillan K, Quirke P. Unlocking the archive--gene expression in paraffin-embedded tissue. J Pathol. 2001;195:66–71. doi: 10.1002/1096-9896(200109)195:1<66::AID-PATH921>3.0.CO;2-F. - DOI - PubMed
1. Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002;161:1961–1971. - PMC - PubMed
1. Cronin M, Pho M, Dutta D, Stephans JC, Shak S, Kiefer MC, Esteban JM, Baker JB. Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol. 2004;164:35–42. - PMC - PubMed
1. Haselkorn R, Doty P. The reaction of formaldehyde with polynucleotides. J Biol Chem. 1961;236:2738–2745. - PubMed
1. Chaw YF, Crane LE, Lange P, Shapiro R. Isolation and identification of cross-links from formaldehyde-treated nucleic acids. Biochemistry. 1980;19:5525–5531. doi: 10.1021/bi00565a010. - DOI - PubMed

Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials - PubMed (original) (raw)