Straightforward and Precise

Precise measurement of RNA using DireCtQuant100ST

Introduction:

The analysis of biological samples is usually performed by multistep procedures consisting of sample extraction, single or multistep purification and subsequent analysis. All this steps are associated with inevitable sample loss and introduction of technical bias. As the number of steps in given protocol grows, more technical bias is introduced and less accurate and reproducible results are obtained. Moreover multistep procedures are associated with higher cost and longer time to complete.

Objective:

To estimate the reproducibility of the RNA measurement in 4 samples prepared with DireCtQuant100ST solubilisation reagent.

Methodology:

Overnight culture of yeast (Saccharomyces cerevisiae) was grown in YPD media (10 g/l Yeast extract, 20 g/l Bacto peptone, 20 g/ Glucose). The yeast concentration was determined using hemocytometer . Four 250 ul aliquots containg 106 yeast cells each ware centrifuged (1000 g 10 min) and the media was discarded. The pellets were re-suspended in 1ml DireCtQuant100ST DNA/RNA/Protein solubilization buffer. The resulting suspensions were incubated at 90ºC for 3min with shaking (750rpm). A colour shift of the sample from red to orange was observed. The samples were left for 5 min to reach room temperature and centrifuged at 10.000g for 10min. The supernatant was transferred to a fresh tube. Detection of the 18S rRNA was performed using single step RT-qPCR kit. 1ul sample was used in 20ul (final volume) reaction. This amount of sample corresponds to just 1000 individual yeast cells.

Rezults:

Robust detection of the 18S rRNA was observed at Cq 17 with very high precision. The observed standard deviation was 0.07 cycles, this corresponds to 5%. A clear, single pick was observed during melting curve analysis (Fig1).

Conclusions:

RNA was successfully detected with exceptionally high reproducibility staring from just 1000 yeast cells. The described single step, single tube procedure which requires no specific equipment is giving analysis ready sample in less than 10min. The obtained sample from a 0,25ml culture (around 106 cells) in theory could be enough for the analysis of 1000 genes. As additional advantage detection of DNA by qPCR and proteins by Western Blot could be performed using the remaining more than 99% of the sample. The sample could be reliably conserved for a long periods (months at -20ºC) and additional analysis performed as required.

Fig1. RT-qPCR amplification plot of 18S rRNA from Saccharomyces cerevisiae extracted from 1000 cells. The red outline insert demonstrates exceptional reproducibility of the Cq observed at just 0.07 cycles. Black outline insert demonstrates melting curve analysis of the amplified product. Single melting pick with SD less than 0.4ºC is observed.