Formalin fixation impairs RNA integrity in a time dependent manner.

<p>Multiple aliquots of a human liver sample were fixed for different time periods ranging from 4 hr to 120 hr in formalin before paraffin embedding. (A) Similar RIN values were obtained for all fixation timepoints ranging from 2.1 to 2.7. (B) qRT-PCR amplification of fragments of different length in the GAPDH assay reveals a rise in ct values of all amplicons correlating with the fixation time. Prolonged fixation induces a steeper slope of the generated curves indicating increased fragmentation and makes the amplification of fragments longer than 277 bp impossible. (C) The ct values obtained for the 277 bp amplicon are correlating with the fixation time. qRT-PCR was performed in triplicates. Graphs depict median ct of triplicate qRT-PCR analyses, error bars depict standard deviation.</p

Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

<div><p>Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples.</p></div

Comparison of RIN values and qRT-PCR results of different FFPE and cryopreserved samples.

<p>RIN and ct-values generated by qRT-PCR of RNA extracted from 65 different samples of 15 patients are shown. Corresponding aliquots of different tissue types were (A) cryopreserved or (B) fixed in formalin and embedded in paraffin. The majority of cryopreserved samples have RIN values above 5 while the majority of FFPE samples show low RIN values between 2 and 3. 11 FFPE samples showed high RIN values above 3 and up to 8 but ct values of the qRT-PCR assays of these samples were not substantially lower than in the other FFPE samples. In CRYO samples the Pearson correlation factor between RIN and ct values is −0.5 to −0.6, in FFPE samples the correlation factor is −0.25 to −0.28 depending on amplicon length. The difference in ct values obtained from CRYO or FFPE samples is statistically significant (p<0.01). PCR was performed in triplicates and median ct is shown. Samples not amplifying or yielding unspecific products were set to a ct of 40 (but omitted from statistical analysis).</p

Fixation introduces a bias towards small RNA species in qRT-PCR.

<p>(A) Small RNA fragments like 5 s rRNA (∼120 bp) are present in cryopreserved (left) and fixed (right) samples of human liver. (B) Longer RNA fragments including 18 s (1,9 kb) and 28 s (5 kb) show an increase of 2 or 3 cycles in TFPE and FFPE samples respectively, revealing a stronger impact of fixation on larger RNA fragments. Bar graphs depict median of qRT-PCR triplicates and standard deviation.</p

Fixation has strong influence on RNA fragment size and impairs qRT-PCR results.

<p>Electropherograms show different RNA molecule size distribution in cryopreserved and FFPE/TFPE liver tissue samples. (A) In contrast to CRYO, ribosomal RNA peaks are absent and fragmentation of RNA has occurred in all fixed tissues. (B) RNA cleanup by Qiagen RNeasy Kit removes small (<200 bp) RNA molecules. (C) RNA extraction using the RNeasy miRNA kit (Qiagen) enriches for small RNA fragments. (D) Extraction with RNeasy FFPE kit (Qiagen) does not change the overall size distribution of fragments but also enriches small molecules. Purification on column based systems (B–D) yields higher 260/280 ratio indicating less contamination of RNA. (E) Fixation has strong impact on the raw ct values and standard deviation of triplicates in qRT-PCR of three different housekeeping genes in cryopreserved, FFPE and TFPE tissue. Note that fixation markedly changes the ratio of ß2 microglobin (B2M) to GAPDH and this effect is further influenced by RNA isolation method used.</p

Fixation and storage introduces major gene-to-gene variations in qRT-PCR.

<p>Aliquots of a human liver sample were cryopreserved or fixed in formalin and paraffin embedded. (A) RNA was extracted from samples at different timepoints including technical replicates. (B) Comparison of qRT-PCR data for 92 genes from cryopreserved and FFPE human liver samples reveals an average difference of the ct values ranging from 4 cycles (6 months) to 8 cycles (1 year) increasing with storage time at room temperature. Extraction from the FFPE sample which had been stored for one year was done in duplicates. cDNA generation was performed in duplicates from the same RNA, and qRT-PCR was performed twice from the same cDNA. Data was generated with the TaqMan “Human Molecular Mechanisms of Cancer” assay, individual ct values are shown.</p

Impact of fixation on cDNA synthesis efficiency.

<p>(A) cDNA generation from RNA extracted from cryopreserved and TFPE human liver tissue is in direct correlation to the amount of template RNA. RNA extracted from FFPE tissue produces only small amounts of cDNA even when more template RNA is provided. cDNA from (A) was tested in the amplicon length qRT-PCR assay. (B) Ct values from the cryopreserved sample are in the range of 22–25cts. Ct values of TFPE samples are shifted by approximately 4 cycles but the appearance of the curve remains similar to that obtained from cryopreserved samples. In FFPE tissue the longer amplicons are not amplified, and even for the short amplicons the ct values are very high (31–36ct). Error bars in A depict standard deviation of the median qRT-PCR result in three individual cDNA preparations, or in B the standard deviation of three qRT-PCR analyses. RIN values and 260/280 ratios obtained from different samples: CRYO RIN: 7,7; 260/280: 1,97; TFPE RIN: 2,5; 260/280: 1,98; FFPE RIN: 2,7; 260/280: 2,01.</p

Assay for evaluation of RNA performance in qRT-PCR.

<p>qRT-of six amplicons of different length (71, 153, 200, 277, 323, 530 bp amplicon size) the gene GAPDH performed using cDNA derived from FFPE, TFPE and cryopreserved samples of different organs. (A) Gel images of GAPDH amplicons in three different CRYO, FFPE and TFPE tissues. All size fragments can be amplified from CRYO samples, most larger amplicons are missing in FFPE samples, all amplicons are present in TFPE samples but the 530 bp band is weak. (B) Direct comparison of ct values obtained from CRYO, FFPE and TFPE liver tissue. In FFPE samples the 323 and 530 bp bands could not be detected or were unspecific, and ct values of smaller amplicons were shifted by up to 9 cycles. The slope of the regression line through the data points was greater in FFPE (2.14) than in TFPE (0.55) and the cryopreserved (0.34) liver samples. Error bars depict standard deviation of PCR triplicates (too small to be visible in CRYO and TFPE samples).</p

Technical controls performed on mice with and without DDC treatment.

<p>In order to complement the biological controls (knockout mice) we performed a number of technical controls to make sure that no cross reactions between the different reagents might lead to false results.</p

Influence of channel amplification on demonstration of antigen colocalization in <i>is</i>PLA and IF in 12 week-DDC treated <i>krt18^−/−</i> mice.

<p>(<b>A, B, C</b>) Show different levels of digital amplification of the green channel (IF for K8) and constant amplification of the red channel showing <i>is</i>PLA for K8 and p62. Arrows indicate a MDB-like aggregate that was constantly positive for K8 but negative for p62. Arrowheads indicate MDBs which were positive in <i>is</i>PLA for p62 and K8 but IF showed only presence of K8 (yellow merged signal) in <i>is</i>PLA positive MDBs after maximal amplification of the green channel. Scale bars: 20 µm.</p