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Fast Reverse Transcription from Mouse Blood Samples using PrimeScript Reverse Transcriptase

TECH NOTE

Fast Reverse Transcription from Mouse Blood Samples using PrimeScript Reverse Transcriptase

Data kindly provided by: Philip H. Jones and Chioma Okeoma, University of Iowa

Overview

The PrimeScript RT Reagent Kit (Perfect Real Time) is a simple and fast reagent for reverse transcription—cDNA templates for quantitative PCR (qPCR) can be prepared in around 15 minutes. In this experiment PrimeScript Reverse Transcriptase was used to generate cDNA from mouse peripheral blood mononuclear cells (PBMCs) and serum. The resulting cDNA was then used to probe the expression of a target gene using qPCR. This experiment shows that PrimeScript RT can quickly generate qPCR-ready cDNA using a streamlined protocol.

Results

The PrimeScript RT kit generated cDNA ready for qPCR with just a 15 minute amplification step. qPCR of the synthesized cDNA showed that the relative expression levels of the target gene were higher in infected BST2(-/-) mice compared to wild-type mice in both serum and PBMC samples. The difference in the relative expression level of the target gene between infected BST2(-/-) and wild-type mice was greater in serum samples than in PBMC samples.

Difference in target gene expression levels in wild type and BST2(-/-) mice. Relative expression levels of the target gene as determined by qPCR. The fold increase in expression of the target gene in BST2(-/-) mice is shown for PBMC (Panel A) and serum (Panel B) samples.

Conclusion

The PrimeScript RT Reagent Kit (Perfect Real Time) was able to generate cDNA from total RNA isolated from mouse PBMC and serum samples in ~15 minutes. The cDNA was used to confirm higher expression levels of the target gene in infected BST2(-/-) mice by qPCR.

Methods

Blood was collected from the retro-orbital sinus of individual infected or uninfected wild-type or BST2(-/-) mice (strain background: C57BL/6) in heparinized capillary tubes. RNA was isolated from PBMCs and serum using an RNeasy Mini Kit (Qiagen). RNA was diluted to a final concentration of 37 ng/µl and 5 ng/µl for PBMC and serum samples, respectively. The reverse transcription reaction was prepared according to the PrimeScript RT Reagent Kit (Perfect Real Time) user manual. The reactions were incubated at 37°C for 15 minutes, 85°C for 5 seconds, and cooled to 4°C in a Veriti Thermal Cycler (Life Technologies). Two microliters of the reverse transcription reaction was used for qPCR with the SYBR® Premix Ex Taq (Tli RNase H Plus) kit, according to the recommended protocol, using a 7500 Fast Real-Time PCR System (Life Technologies).

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