I was hoping to get some input regarding the process I use to extract P. falciparum mRNA from dried blood spots for use in qPCR.
I am trying to amplify Pfs25 mRNA, and I've been running into two major problems:
1. Issues with RNA purity
-overall concentrations are low, about 20-80 ng/uL according to NanoDrop
-260/280 values average around 1.5
-260/230 values average around 0.5
2. gDNA contamination of RNA samples
-no reverse transcriptase control in qPCR consistently shows amplification with Ct values close to the experimental wells
I use Qiagen buffer AL, buffer ATL, and proteinase K (DNA mini kit) to create the lysate from the dried blood spot, and then I use the Qiagen RNeasy mini kit to extract from that prepared lysate. I synthesize cDNA using the Qiagen Quantitect Reverse Transcription kit, and I do include the wipeout buffer step.
Has anyone else run into these problems while extracting RNA from dried blood spots? I've tried quite a bit of generic qPCR troubleshooting, and none of it has seemed to make any significant difference.
Thank you in advance for any advice you might be able to give me!
Try to maximize the purity of your RNA before you start RT-PCR.
# use DNase to degrade your contaminated DNA.
# take enough starting material to increase the concentration of your RNA
# Finding the most appropriate method of cell or tissue disruption for your specific starting material is important for maximizing the yield and quality of your RNA preparation
# Resuspend the purified RNA pellet. After preparing an RNA sample, it is crucial that RNA be suspended and stored in a safe, RNase-free environment.
#store RNA at –80°C in single-use aliquots, resuspended in one of several RNA storage solutions designed for this purpose:
#THE RNA Storage Solution (1 mM sodium citrate, pH 6.4 ± 0.2)
#0.1 mM EDTA (in DEPC-treated ultrapure water)
#TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0)
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