Assessment of nucleic acids extraction kits
We compared the quality and quantity of nucleic acid extraction in five commercially available extraction kits, using prostate needle biopsies with normal histology, assuming that normal tissue is less heterogeneous than malignant tissue, thus reducing the risk of biased comparison. We found that RNA extraction with RNeasy® had higher yields of RNA, purity (A260/A280) and RIN-values. Furthermore, the agreement in RNA yield and quality (A260/A280 and RIN-values) between the two compared kits was low, with consistently lower yields and purities in the High Pure kit. RNA of high quality is essential when performing molecular studies, and thus even small differences in purity and RIN-values are important to consider when choosing extraction kit. Based on the results in this trial, the RNeasy® kit would be preferred for extraction of high quality RNA from FFPE prostate biopsies.
In a second trial, the RNeasy® FFPE kit was compared to the AllPrep® DNA/RNA FFPE kit. The two kits produced similar yields and purity of the extracted RNA, however, higher RIN-values were found in samples extracted with the RNeasy® kit. Even though it would typically be preferable to extract both DNA and RNA from the same tissue section, the consistently lower quality of RNA produced with the AllPrep® kit suggests that using two separate extraction kits for DNA and RNA would be preferable if high quality RNA is desired.
A possible explanation for the kit-to-kit variations in RNA quantity and quality could be the conditions recommended for the proteinase K digestion. The use of proteinase K digestion is necessary to release RNA from the meshwork of cross-linked proteins and nucleic acids, and it has also been shown to play an important role in the extraction of longer stretches of nucleic acids (>200 bp) [
14,
19,
20]. However, the proteinase K digestion does not attack the methylene bridges that forms the crosslinks [
3]. The RNeasy® kit uses a proteinase K treatment for 15 min at 56 °C followed by 15 min at 80 °C, the latter being critical for reversal of crosslinks introduced by formalin fixation. The High Pure kit on the other hand recommends a proteinase K digestion for 3 h at 55 °C. It is therefore possible that the digestion process in the High Pure protocol is less efficient in breaking the crosslinks in the tissue, thus resulting in less RNA yield, which is also more fragmented than samples extracted with the RNeasy® kit.
In a third trial, we compared the two DNA extraction kits QIAamp® DNA FFPE Tissue kit and High Pure FFPET DNA Isolation kit, and found similar DNA yields, although with higher purity (A260/A280) in the QIAamp® kit. One sample extracted with the QIAamp® kit had a high A260/A280 ratio (2.66), which is probably due to a RNA contamination within this sample. Based on these data, we chose the QIAamp® kit for comparison with the AllPrep® DNA/RNA FFPE kit. In this final trial, similar DNA yields in samples extracted with the two kits were found, although once again with higher purity (A260/A280) in the QIAamp®. Thus, our analyses suggest the QIAamp® kit as the best performing DNA extraction kit from FFPE prostate biopsies.
Assessment of DNA/RNA quantity and quality from archival biopsies
Performing molecular studies on archival FFPE needle biopsy material have potential limitations. Prostate needle biopsies are often performed using an 18-gauge needle, yielding a biopsy which is roughly 1 mm in diameter and 12–15 mm long. Sectioning such a biopsy would in theory yield 100 sections of 10 μm. However, the biopsy has typically been sectioned previously as a part of clinical evaluation, and thus only a limited amount of tissue with representative disease histology is available for research studies. Furthermore, according to Swedish laws one must make sure that there is always representative cancer tissue left after sections are taken for research studies. Thus, in practice, it might be possible to take four to five 10 μm sections for research studies. The aim of this second part of the study was thus to investigate if it is possible to extract nucleic acids from archival FFPE prostate biopsies using one 10 μm section of tissue. We also wanted to investigate the quantity and quality of the nucleic acids extracted from the biopsies in order to investigate which type of molecular studies could be performed.
Based on the results from the assessment of nucleic acid extraction kits, nucleic acids were extracted using the QIAamp® DNA FFPE Tissue kit and RNeasy® FFPE kit. DNA was successfully extracted from all 84 biopsies with a mean yield of 3.7 ng/μl and a purity of 1.7. One DNA extract had a high A260/A280 ratio (5.3), indicating a RNA contamination of this sample. RNA was also successfully extracted from all 84 samples, with a mean yield of 10.6 ng/μl. The purity of the extracted RNA had a mean value of 1.55 and the RIN-value a mean of 2.3. The expression of 18S5 RNA showed high variability between samples, indicating that this is not an optimal endogenous control gene for prostate tissues. The amount of tumor (in millimeters) had the strongest association with both quantity and quality of the nucleic acids extracted from the biopsies within this study, which is consistent with a previous study showing an association between tumor amount, measured by millimeter tumor of a renal biopsy, and purity of RNA (A260/A280) [
21]. Our results indicated no correlation between age of the FFPE specimen and neither RNA yield or integrity of the extracted RNA. This result has been supported by several previous studies [
10,
21,
22], although others have seen an association between age of the tissue and RNA quality, but not yield [
23]. However, we did find a correlation between age of the FFPE specimen and DNA yield in the present study, which has also been shown previously by Carrick et al.
, who observed a significant association between DNA yield and quality and storage time [
24]. Nucleic acids extracted from FFPE samples are generally more degraded than samples from fresh frozen material, resulting in reduced qPCR amplification of DNA and RNA targets larger than 200 base pairs (bp) [
25‐
27]. In the present study, a 90 bp amplicon of 18S5 rRNA was amplified with success (C
T < 38) in 83 of 84 (98.8%) RNA samples and 81 of 84 (96.4%) DNA samples. The RNA sample that was not amplified had the lowest purity of all samples (A260/A280 = 0.7), however for the DNA samples there was no apparent reason to why the amplification failed. These results indicate that archival FFPE prostate biopsy tissues are suitable for PCR amplification even though the small quantity and poor quality of the isolated nucleic acids.
In order to gain new insights to pathological processes, quantitative measures of mRNA levels and DNA analyses investigating for example mutations, polymorphisms and epigenetic changes have become essential. It is recommended that only high-quality DNA and RNA are used for these analyses. In some instances, however, such as in studies of biomarkers of prostate cancer mortality, high quality samples may not be available. Typically using low-quality samples introduces bias toward the null, because the degradation of samples entails random variability that is similar across comparison groups. Therefore, precision and power is impaired, and the risk of false negative associations, but not false positive, increases.
Many studies have investigated the effect of poor RNA quality (RIN-value) and DNA quality, using techniques such as qPCR, microarrays and NGS [
10,
12,
23,
24,
26,
28‐
35]. RIN-values are considered the most powerful predictor of microarray quality [
30]. Although moderately degraded RNA could yield acceptable microarray results, extensively degraded samples such as those obtained in the present study, should usually be excluded from analyses. Only samples with RIN-values ≥7 are normally recommended for microarray studies [
36]. However, the cDNA-mediated Annealing, Selection, extension and Ligation (DASL) assay have been shown to tolerate input RNA degraded to an average of 100–200 bp, while still yielding robust results [
37]. This technology has successfully been used to profile archival FFPE prostatic tissues previously, including by our group [
38‐
41]. Achieving information on single nucleotide polymorphisms (SNPs) in FFPE DNA has been an issue due to low quantity and quality of the DNA. However, studies comparing results obtained from fresh frozen and FFPE tissues have shown a high concordance for some SNP arrays [
42,
43], and new platforms have allowed generation of reliable results even from small quantities of FFPE DNA [
44]. NGS is another technology used for both large-scale and targeted analysis of both DNA and RNA samples, and it has been shown that the success rate of the technique is not associated with storage time of the FFPE tissue, not even for samples stored up to 32 years [
24]. Furthermore, a good correlation of NGS data has been found between fresh frozen and FFPE tissues, even though a higher sequencing coverage (× 80) is recommended for FFPE tissues [
23,
31‐
35]. Measuring gene expression levels in FFPE samples using qPCR has been suggested to be more robust than through microarrays [
26]. This suggestion is strengthened by findings showing that RIN-values cannot be used in order to predict performance of the qPCR [
10,
26,
45,
46]. Amplification of products > 400 bp using qPCR is strongly dependent on good RNA quality (RIN > 5), although if smaller amplicons (70–250 bp) are investigated, the results are more or less independent of RNA quality [
12]. In the past it has been problematic to perform large scale qPCR analyses on FFPE samples due to high C
T-values and limited concentrations of the extracts [
47,
48], however techniques such as TaqMan® PreAmp has addressed this challenge faced by researchers working with small samples such as needle biopsies, from which only limited amounts of RNA could be extracted. The simple process includes pre-amplification of as little as one ng of cDNA, and enables the user to perform qPCR amplification of up to 384 target genes per pre-amplification reaction on TaqMan® low-density arrays [
49]. Targeted qPCR and pyrosequencing can also be used to provide reliable data when analyzing FFPE DNA samples [
50], and the methods can overcome the limits of using FFPE DNA by designing assays specific for small DNA fragments and improving the PCR protocol for low DNA inputs [
51,
52].
An advantage with our study is that the extraction kits have been compared using serial sections of the same biopsies, which decreases the risk of differences between the kits due to variations in the tissue histology. Furthermore, the extraction kits were tested on biopsies with normal histology, which further decreases tissue heterogeneity. The best performing extraction kits for DNA and RNA was subsequently used for extraction of nucleic acids on 84 biopsies with malignant histology, yielding similar quantities and quality of the nucleic acids as in the normal biopsies. The study has a number of limitations. 1) The limited number of nucleic acid extraction kits tested. Many other kits for extraction of nucleic acids from FFPE tissues exist on the market, with the potential to perform better than the kits used in this study. 2) The small number of comparisons performed for each extraction kit (
n = 10). Further studies on larger tissue materials are needed in order to validate the results found within the present study. 3) Even though the tissue sections used for comparisons of extraction kits were cut immediately adjacent to one another, there could still have been variations in the tissue area that influenced the DNA/RNA yield. 4) Due to the limited amount of tissue available in a prostate biopsy, extractions could not be performed in duplicates from the same biopsy. Furthermore, two different operators performed the extraction of nucleic acids with the kits to be compared, which could have affected the outcome due to inter-operator variability [
15].