Tissue Collection - Library Preparation
Fresh tissue to cDNA Libraries
When selecting the tissue it is important to be consistent in your sampling of different individuals/treatments/locations. Keep in mind that gene expression and SNP detection will be affected by both environmental conditions and tissue type. Before starting the lab work, it is important to note that RNA is highly susceptible to degradation by RNAse enzymes. These enzymes can be found in the air and on your clothing and hands. It is very important to utilize good laboratory practices and work as efficiently as possible through cDNA synthesis. Once you have double stranded cDNA you can breathe a little easier, because cDNA is more stable than RNA. While there are many RNA extraction kits to choose from we recommend using Qiagen's RNeasy extraction kit. It is a very fast and easy procedure, free from toxic chemicals. However, if you plan to also extract genomic DNA from your tissue, a Trizol extraction method may be preferable. While there are also many cDNA library preparation kits, Illumina's TruSeq RNA sample preparation kit is all-inclusive. It includes all the necessary reagents to purify mRNA from total RNA and make cDNA libraries. The protocol is straightforward and there is peace of mind knowing the manual was written and tested by the manufacturers of the Illumina sequencing machine.
Collins L, Biggs P, Voelckel, C, Joly, S. 2008. An approach to transcriptome analysis of non-model organisms using short-read sequences. Genome Informatics 21: 3-14.
RNA extraction kits and protocols
Quantification/validation of samples
Library Prep/RNA seq technology
2. Quantify the amount of total RNA in each sample using a QuBit RNA assay. (http://products.invitrogen.com/ivgn/product/Q32852).
library preparation. 1 µg of total RNA is often a good amount to start with.
SummaryYou have now extracted total RNA from a tissue, purified mRNA from the total RNA, and from this mRNA have created cDNA libraries that are ready for sequencing. Congratulations! You are well on your way to collecting a single data set that contains both gene expression and SNP data from the tissue you have selected.
De Wit P, Pespeni MH, Ladner JT, Barshis DJ, Seneca F, Jaris H, Overgaard Therkildsen N, Morikawa M and Palumbi SR (2012) The simple fool's guide to population genomics via RNA-Seq: an introduction to high-throughput sequencing data analysis. Molecular Ecology Resources 12, 1058-1067.