Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

Background Since the emergence of next generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA extraction methods using whole blood sampled under varying conditions from 20 wild carnivores. Results Despite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 – 18.4 μg) with varying integrity (RIN 4.6 - 7.7), the latter being significantly affected by the storage and extraction method used. We observed a significant overall effect of the extraction method on DNA contamination. One particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if libraries were not depleted of hemoglobin prior to sequencing. Conclusion By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species.

Three µl RNA of each wolf sample were reverse-transcribed following the manufacturers' protocol (Mint cDNA synthesis Kit, Evrogen). We determined the optimal numbers of cycles of the 2 nd strand synthesis for each sample individually. This is required because PCR overcycling of 1 st strand cDNA yields nonspecific PCR products whereas too few cycles result in low yields. Both are undesirable for high-throughput applications such as next generation sequencing applications. After the evaluative PCR, the 1 st strand cDNA obtained from the total RNA sub-sample of the Artic wolf (sample A2) was further amplified with 18 additional cycles on the thermal cycler whereas all remaining sub-samples were subjected to additional 17 PCR cycles.
To remove remaining primer excess, buffers and the enzymes from 1 st strand cDNA products, the cDNA was run on a 0.8% low melting point agarose gel and extracted using Zymoclean Gel DNA recovery Kit (Zymo Research). During this process we also removed fragments of 750 bp length for all samples, potentially representing hemoglobin transcripts. A total of three purified extracts (A1, B1 and B2, ", Table 2) were used for normalization of the cDNA (Trimmer Kit, Evrogen). The normalization of full-length-enriched cDNA libraries (NORM, Table 2) is based upon the selective ability of the enzyme duplex-specific nuclease (DSN) to preferably cut double stranded DNA.
Very abundant transcripts will be degraded until only an equalized single-stranded cDNA fraction will remain intact [1,2]. This procedure is supposed to allow a more sufficient detection of low abundant transcripts. The ¼ dilution of DSN provided the best results for all experimental samples. After normalization all three samples were subjected to 13 PCR cycles for 1 st amplification. The full-length-cDNA libraries were purified using the gel extraction procedure as described above followed by 12 PCR cycles for a second amplification. The second amplification step became necessary to increase the proportion of long transcripts in the cDNA sample. The process of PCR tends to favor the amplification of smaller products over larger ones, which can result in the loss of rare, long transcripts. Using specific primers provided with the kit we were able to ensure that longer transcripts were amplified more effectively than smaller ones.

Sequencing library preparation and 454 sequencing
Novel pyrosequencing techniques require specific library preparation steps in order to facilitate the fragment size suitable for the respective method, the ligation of adapters and an accurate quantification of the copy number prevalent in the final library.
We started the 454 GS FLX Titanium library preparation from the gel-extracted cDNAs and skipped the nebulization step as visual inspection of the agarose gel revealed only a small proportion of fragments being longer than 1,000 bp. Since we were interested in the full spectra of mRNA transcripts, we also did not remove small fragments from the cDNA pool. The samples were further subjected to blunt ending, 454 Titanium adapter ligation, various purification steps and a final release of single-stranded DNA fragments from the capture beads. This resulted in 30 µl of single stranded DNA library and to quantify the yield we used a procedure suggested in [3] which relies upon the efficiency of quantitative PCR. As suggested [3], we established a DNA standard using an old Titanium library, which was amplified using the 454 GS FLX Titanium emPCR priming sites as annealing targets and evaluated the copy number via the respective DNA concentration in the purified PCR product. The PCR product was diluted to cover a range from E 02 to E 09 copies. All samples and the standard dilutions were set up for a qPCR on  Total 20 * Blood from one of the individuals has only been collected for the whole blood and not for the LeukoLOCK approach. ** Samples have been collected in a zoo-like facility or sanctuary.