Improved RNA quality and TaqMan®Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials
© Li et al; licensee BioMed Central Ltd. 2008
Received: 26 July 2007
Accepted: 06 February 2008
Published: 06 February 2008
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 from the Bioanalyzer and TaqMan® data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70°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.
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.
Archival Formalin-Fixed, Paraffin-Embedded (FFPE) tissue samples represent a robust and invaluable source of human tissue for gene expression analysis. Compared to fresh and snap frozen tissue, FFPE tissue has an inherent advantage in that retrospective patient data, including survival history and treatment response etc, is readily available, allowing immediate comparison with clinical pathological parameters. Data generated can potentially highlight biomarkers useful in disease classification, diagnosis and prognosis, and potentially elucidate novel therapeutic targets [1, 2].
However, these tissues have not been widely used in molecular biology due to the degradation and chemical modification of RNA extracted from FFPE blocks. RNA extracted from FFPE is degraded to fewer than 300 bases  in length because archived blocks are often stored at room temperature for long periods of time. The situation is made more complicated by the fact that RNA is modified by methylol groups to form cross-links with protein or nucleic acid during formalin fixation [4–6], which results in poor yields [1, 7] and compromised extracts.
Real-time quantitative TaqMan® reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis has been introduced as a sensitive, accurate, and highly reproducible method to study gene expression . It has been successfully used to detect gene transcript levels from snap frozen tissue extracts and even from FFPE containing partially fragmented RNA [9–11] although the detection rate is lower as indicated for example by invariably higher CT values in the latter [12–16].
RNA yields from FFPE materials. Extraction products were assessed spectrophotometrically using NanoDrop® ND-1000 Spectrophotometer. Three extractions were carried out using each protocol.
OD Ratio (260/280)
2.1 +/- 0.1
337 +/- 58
2.1 +/- 0.1
147 +/- 26
1.6 +/- 0.1
186 +/- 67
1.6 +/- 0.1
108 +/- 72
Evaluation of modified protocols
Comparison of TaqMan®Universal PCR Master Mix (UPMM), Gene Expression Master Mix (GEMM) and PreAmp
Evaluation of TaqMan®PreAmp using CDKN1B
Comparison of CTdifference between FFPE and snap frozen cells
RNA analysis has generally been performed on snap-frozen or fresh materials, using variable techniques including microarray, northern blotting and RT-PCR. However, a constant challenge has been robust and reliable analysis of gene expression in archival tissues. This application has been frustrated by poor RNA yields, small sizes of extracted fragments and low levels of detectable RNA in the extracts . In this study we established a cell line model to test modified extraction protocols and quantify differences in performance using a panel of QRT-PCR assays on FFPE compared with parallel snap-frozen cell preparations using TaqMan® methodology. We found that our modified extraction method and TaqMan® PreAmp enhanced expression analysis from FFPE cells optimally.
RNA and FFPE
Generally, RNA extracted from FFPE materials is fragmented and chemically modified. Fragmentation occurs possibly since tissues are surgically removed, and it continuously occurs during fixation and preservation. Experimental design was such that equal numbers of cells were processed for FFPE cell block construction and snap freezing for comparative purposes. By using cells fixed under controlled conditions possible variables due to the effects of storage on RNA degradation were negated, thus revealing the true impact of formalin fixation on RNA quality. We extracted intact RNA fragments in a separate experiment from pellets with a large number of formalin fixed paraffin embedded normal thyroid cells (Figure 4), which was consistent with a finding by Scicchitana et al . In that study, intact RNA molecules were extracted from newly formalin fixed paraffin embedded human bone marrow stromal cells suggesting that RNA degradation could be a minor problem for recently fixed FFPE in some types of cells. Our study showed that the differences in RNA quality between the small and large fixed pellets (Figure 2 and Figure 4) might be due to the fact that the majority of cells in the centre of the large pellet were remained alive during construction of the large solid cell pellet. This could limit the access of RNases to intracellular mRNA because degradation will not fully occur until the fixation process begins resulting to the intact mRNA being maintained. Unfortunately, intact mRNA is not generally obtained in majority of fixed tissues or cell lines, and RNA extracted from FFPE is normally degraded to fewer than 300 bp.
Figure 4 displays the degree of RNA modification in FFPE cells caused by methylol groups during formalin fixation. Masuda and colleagues  suggested that formaldehyde reacts with RNA forming an N-methylol followed by an electrophilic attack to form a methylene bridge between amino groups. Interestingly, our results show a clear large RNA band, approximately 5 kb in length (Figure 4-lane 6), which is hypothetically the cross-linked RNA. The other two bands visualized were also larger than that extracted from snap frozen cells indicating the modification of RNA in FFPE. Furthermore, with incubation at high temperature, the large RNA band (~5 kb) was removed, and the sizes of the other two bands became closer to 18 s and 28 s of the snap frozen extracts. This is in agreement with the results of a mass spectrometric analysis that was carried out by Masuda group, suggesting methylol modification is reversible by heating [18, 19]. However, a balance must be achieved between breaking cross-links while not contributing to degradation of labile RNA. Our results showed that incubation at 70°C for 20 minutes was the optimal condition, among those tested, to de-modify cross-linked RNA while maintaining RNA integrity (Figure 4).
A prerequisite for gene expression studies in formalin-fixed tissue samples was the establishment of a reliable and reproducible extraction method to provide detectable RNAs for subsequent analysis . The quality of extracted RNA can be variable among different extraction methods due to several factors, such as the contamination of RNases, proteins and genomic DNA . Our results showed that proteinase K digestion based protocols followed by on column DNase digestion and RNA elution produced good detectable RNA of FFPE.
Proteinase K digestion has also been demonstrated by many laboratories to be critical in RNA extraction protocol [1, 3, 21, 22] possibly because of two functions. On one hand, it can degrade proteins that are covalently cross-linked with each other and nucleic acid to release RNA from the matrix, thereby allowing efficient RNA extraction from FFPE materials . On the other, it can inactivate RNases that tend to be stable and do not require cofactors to function. Thus its activity avoids any potential reactivation of RNase during reversal of fixation in aqueous buffers .
Our data demonstrates that incubation in proteinase K buffer at 70°C for 20 minutes facilitated the disruption of cross-links, resulting in improved quantity and quality of RNA. The RNA extracted using the modified protocols (with the incubation) enhanced detection by a mean of 3 cycles (Figure 3), which is equivalent to 8 fold increased sensitivity. It is most likely that the additional incubation step removed the remaining cross-links between RNA and protein leading to the longer RNA molecules being extracted . In addition, this incubation denatured proteinase K, thus avoided its further damage to RNA in the following purification procedure, which suggests the necessity of a heating (at 70°C for 20 min) application in any proteinase K based extraction of FFPE samples.
TaqMan® QRT-PCR technique is based on the 5' nuclease activity of Taq DNA polymerase and involves cleavage of a specific fluorogenic hybridization probe that is flanked by PCR primers [24, 25]. Because of the small target size, many laboratories have demonstrated that it is possible to measure gene expression levels using FFPE tissues as a source of mRNA . Still, it seems to be problematic to perform large scale of analysis on FFPE because of the high CTs and limited concentration of extracts [14, 15].
We employed a novel TaqMan® PreAmp technique which we found to be a practical solution to decrease CT values, and in particular suitable in our hands to generate real-time PCR results from limited amounts of input RNA, such as extracted from laser captured microdissected material . The principle of TaqMan® PreAmp technique is to amplify target cDNA prior to real-time TaqMan® PCR analysis. Briefly, cDNA is synthesized from total RNA by use of random priming. The cDNA for the specific target assays is then amplified by pre-amplification reaction using pooled gene-specific primers to increase the number of targeted copies. The pre-amplification product is diluted and finally analyzed by real-time TaqMan® PCR using single assay containing one pair of gene-specific primers and probe.
The TaqMan® PreAmp technique addresses the challenge faced by researchers working with rare or precious samples, which only limited RNA could be extracted from, to perform gene expression analyses using real-time QRT-PCR. The simple process enables the user to perform uniform amplification from as little as one nanogram of cDNA (which was used in this study) or alternatively conduct up to 200 real-time PCR reactions per pre-amplification reaction without compromising the available sample material. Our results demonstrated that TaqMan® PreAmp overcame the difficulties usually caused by low yields of RNA extraction from FFPE.
Influence of TaqMan®Master Mix and amplicon length
This study demonstrated that the sensitivity of TaqMan® detection was influenced by choice of Master Mix and amplicon length in assay design. We evaluated UPMM and GEMM which showed similar sensitivity for short amplicons (less than 90 bp), while GEMM displayed better sensitivity for longer amplicons (over 100 bp) compared with UPMM in parallel snap frozen and FFPE extracts (Figure 5). This effect was dissipated when UPMM was used after pre-amplification, possibly because of the increased copy number of template available or because of the composition of the PreAmp MasterMix which is closer in components to GEMM than UPMM. Reagents can have a significant effect on assay reproducibility  due to some parameters such as different polymerases sensitivity , primer binding efficiency and the concentration of Mg2+ . Karrer et al. described the Monte Carlo effect using plant material suggesting that the PCR reproducibility could be limited when the number of available templates is low. Increased concentrations of Mg2+ reduced PCR variation possibly by allowing a higher proportion of annealed primers extended by the more active polymerase .
Our data corroborated the observation that amplicon size is crucial in designing assays to analyze gene expression levels not only using FFPE extracts but also using RNAs with high integrity [12, 15]. Many researchers have found that short amplicons generated lower CTs than longer amplicons on analysis of the same gene in FFPE [30, 31]. Data generated in this study evaluating snap frozen samples using two sizes of GAPDH (Figure 3) demonstrated GAPDH-67 generated a reduction in CT by 2 – 3 cycles over GAPDH-122 in both GEMM and PreAmp experiments, demonstrating that smaller amplicons give more consistent results .
In addition, we found CTs between FFPE and snap frozen were closer for small amplicons than that for large amplicons (Figure 7). For example, the analysis of GAPDH using a target amplicon of 67 bp displayed CTs 1 – 2.5 cycles closer between FFPE and snap frozen than the same experiment using the longer amplicon size of 122 bp. We further measured the amplification efficiency associated with these two sizes of GAPDH assays using a broad dilution range (5 Log10s) which showed closed efficiencies with 99.98% for GAPDH-67 and 99.92% for GAPDH-122 (data not shown). This measurement eliminates non-specific amplification that could contribute to decreased amplification efficiency of the true target. Therefore, it seems reasonable to conclude that the shift of efficiency detected from frozen to fixed material (Figure 7) is due to degradation of RNA in FFPE, and logical to extrapolate that the longer an amplicon is, the more likely its template will be degraded in extracted RNA. As a general rule, house-keeping genes or normalising assays should have amplicon sizes that match the size of the target whose expression is to be measured  and amplicons less than 100 bp should be employed in gene expression studies using FFPE materials.
We evaluated the effect of modifying recommended extraction protocols to reproducibly produce RNA that may be successfully amplified using QRT-PCR. We have found the TaqMan® PreAmp system to be a robust and practical solution to limited quantities of RNA and have demonstrated comparable results in matched FFPE and snap frozen preparations providing proof of principle that this method may reliably be utilised in the context of multiple expression analyses from individual FFPE samples.
Cell culture and formalin fixation
Nthy-ori 3-1 (ECACC, Wiltshire, UK) is a normal thyroid follicular epithelial cell line derived from adult thyroid tissue that has been transfected with a plasmid encoding for the SV40 large T gene .
This cell line was grown to confluence in a humidified atmosphere containing 5% CO2 at 37°C in the following plating medium: RPMI 1640 with 2 mM L-glutamine, 10% Foetal calf serum (FCS), Penicillin (100 U/ml) and Streptomycin (100 μg/ml). Tripsinized cells were counted with a hemocytometer. Approximately 1 × 105 suspended cells were aliquot and were pelleted (a) snap frozen and (b) formalin fixed and paraffin embedded into a cell block. When formalin fixation was required, a cohesive solid cell pellet was constructed using 20% agar . The cells were centrifuged in an eppendorf tube, and the supernatant was removed using a pipette. Approximately 30 μl of pre-warmed agar (60°C) was added to each tube. The solid cell pellet was formed within a few seconds. Cell blocks were placed in 10% buffered formalin at room temperature for 5 hours followed by tissue processing on a Tissue-Tek® V.I.P.TM tissue processor for 8 hours comprising: 10% buffered formalin fixation (4 hours at 37°C), 60% ethanol (20 minutes at 37°C), 80% ethanol (20 minutes at 37°C), 100% ethanol (20 minutes at 37°C), xylene (40 minutes at 37°C) and paraffin (80 minutes at 60°C). The pellets were subsequently paraffin embedded.
RNA extraction protocols
Overview of the RNA extraction protocols. Four RNA extraction protocols are Ambion RecoverAll™ Total Nucleic Acid Isolation Kit, Stratagene Absolutely RNA® FFPE Kit, Gentra Purescript® RNA Purification Kit and Invitrogen Trizol® Reagent. (RT. = Room temperature)
50°C for 3 min
Deparaffin – Reagent
RT. for 10 min
RT. for 5 min
RT. for 5 min
Proteinase K digestion
4 μl Protease in 400 μl digestion buffer (final concentration unknown)
50°C for 3 hours
10 μl × 20 mg/ml proteinaseK in 100 μl digestion buffer (final concentration 1.82 μg/ul)
1.5 μl × 20 mg/ml proteinaseK in 300 μl cell lysis solution (final concentration 0.10 μg/ul)
Add Isolation Additive
Pass the mixture through a Filter Cartridge
Pass the mixture through a prefilter spin cup
Pass the mixture through a RNA binding cup
Add protein-DNA precipitation solution
Separate the supernatant
Add Isopropanol and Glycogen
-20°C for 1 hour
Pellet RNA and Ethanol wash
Homogenize in 1 ml Trizol Reagent
Separate the aqueous phase
Add Isopropanol and Glycogen
RT. for 10 min
Pellet RNA and Ethanol wash
Elution buffer 30 μl × 2
Elution buffer 30 μl
RNA hydration solution 30 μl
H2O 30 μl
RNA quantity was assessed using the NanoDrop® ND-1000 Spectrophotometer (Wilmington, USA), and the quality was measured using the RNA 6000 Pico LabChip® Kit on an Agilent 2100 Bioanalyser (Agilent technologies, Waldbronn, Germany).
TaqMan®gene expression assays
Eight of TaqMan® Gene Expression Assays. Seven of these assays were inventoried by Applied Biosystems (P/N: 4331182, Applied Biosystems, CA, USA) and one was a designed assay (GAPDH-67 Forward primer: CAT CCA TGA CAA CTT TGG TAT CGT; Reverse primer: GGG TGG CAG TGA TGG CAT; Probe: ACT CAT GAC CAC AGT CC).
Gene Symbol – Amplicon Length
MT4 – 62
GAPDH – 67
CDKN1B – 71
cyclin-dependent kinase inhibitor 1B (p27, Kip1)
MAPK4 – 72
mitogen-activated protein kinase 4
CD44 – 86
CD44 molecule (Indian blood group)
SDC2 – 103
syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan)
GAPDH – 122
HLA-A – 164
major histocompatibility complex, class I, A
Reverse transcription (RT)
Applied Biosystems High-Capacity cDNA Archive Kit (P/N: 4322171, Applied Biosystems) was used following manufacturer's protocol for reverse transcription. Each RT reaction contained 50 μl of 8 ng/μl total RNA, 10 μl of 10× RT buffer, 4 μl of 25× dNTP mixture, 10 μl of 10× Random Primers, 5 μl of MultiScribe RT (50 U/μl) and 21 μl of RNase-free water. The 100 μl reactions were incubated in an Applied Biosystems Thermocycler for 10 min at 25°C, 2 hours at 37°C and then held at 4°C.
TaqMan®real time PCR
For the Real-time PCR step, amplification was carried out on the Applied Biosystems 7000 Sequence Detection System. Two types of TaqMan® Master Mix were employed in this procedure: TaqMan® Universal PCR Master Mix with UNG (P/N 4304437, Applied Biosystems) or TaqMan® Gene Expression Master Mix (P/N 4370048, Applied Biosystems). The 20 μl PCR reaction included 10 μl of 2× TaqMan® Master Mix, 5 μl of 4× TaqMan® Gene expression Assay (P/N 4331182, Applied Biosystems) and 5 μl of cDNA (RT product 4 ng/μl). The reactions were incubated in a 96-well optical plate at 50°C for 2 min, at 95°C for 10 min, following by 40 cycles of 95°C for 15 seconds and 60°C for 1 min. The real-time PCRs for each assay were run in triplicate.
TaqMan®real time PCR with PreAmp
The preamplification was performed using TaqMan® PreAmp Master Mix Kit protocol (P/N 4366128, Applied Biosystems). The pooled assay mix was prepared by combining 8 of 20× TaqMan® Gene Expression Assays into a single tube and using 1× TE buffer to dilute the pooled assays to a final concentration of 0.2×. The 40 μl of preamplification reaction included 20 μl of 2× TaqMan® PreAmp Master Mix, 10 μl of 0.2× pooled assay mix and 10 μl of 4 ng/μl cDNA sample. The reactions were incubated in an Applied Biosystems Thermocycler for 10 min at 95°C following by 10 cycles of 95°C for 15 seconds and 60°C for 4 min and then held at 4°C. The concentration of the preamplification product was 210 ng/μl (in theory) which was then 1:5 diluted and analyzed by TaqMan® real time PCR using TaqMan® Universal PCR Master Mix following the procedures described.
ΔCT (A-B) = CT X_mean(A) - CT X_mean(B)
Theoretical ΔCT = Log2(Amount cDNA in A/Amount of cDNA in B)
This work was part-funded by Applied Biosystems and received technical support from the company. We would also like to thank staff in the Central Pathology Laboratory, St. James's Hospital, for assistance with fixation and processing of cell blocks.
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