DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate.

Abstract

Using current mRNA quantification methods such as RT-qPCR and RNA-Seq, it is very difficult to examine thousands of tissue samples due to cost and labor of RNA extraction and quantification steps. Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. We showed that appreciate concentration of DTT prevented RNA degradation but not RT in the lysates of several plants tissues, yeast, and zebrafish larvae. Using the buffer, direct reverse transcription on the lysates could produce comparable amount of cDNA with that synthesized from purified RNA. Furthermore, we established DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq) method. DeLTa-Seq is a cost-effective, high-throughput and highly-precise quantification method for the expressions of hundreds of genes. It enables us to conduct large-scale studies using thousands of samples such as chemical screening, field experiments and studies focusing on individual variability.