Ribopure Blood RNA Isolation Kit by Thermo Fisher was utilized to extract the RNA (including small RNAs) as suggested protocol by manufacturer protocol.
Label
FAM-NFQ
Label protocol
A TaqMan Custom RT pool primer (Cat#A25630) of 112 miRNAs was used for cDNA synthesis by reverse transcription. Reverse transcription of mature miRNAs was carried out by using a TaqMan miRNA Reverse Transcription kit reagents (Cat# 4366597) from Thermo Fischer, USA. In short, the reaction mixture contained MultiScribe reverse transcriptase 75 units, RT buffer 1X, OpenArray Custom RT primers 1X, RNase inhibitor 2 units, 3mM MgCl2, 2.0mM deoxynucleotide triphosphates (dNTPs) and 100ng of RNA input limiting 7.5µl the final reaction volume. Thermal cycling of Reverse transcription process was repeated for additional 39 cycles of 16°C for 2 minutes, 42°C for 1 minute and 50°C for 1 second. . In Preamplification, a total 25 µl reaction volume including 2.5µl custom preamp primer pools mixed with 12.5µl of preamp master mix and 2.5µl cDNA and additional nuclease-free water add to reach the final volume. The thermal cycling condition used for preamplification were 95°C for 10 min, 55°C for 2 min, 72°C for 2 min and 12 cycles of 95°C/15 sec, 60°C/4 min. At the end of cycling, preamplification products were incubated at 99.90C for 10 min. for inactivation of enzymes. Preamplification products were diluted (1 to 40) in 0.1X Tris-EDTA buffer. The diluted preamplification products mixed with 2x TaqMan OpenArray Real-Time PCR Master Mix (Cat#4462159) and pipette on OpenArray plates by OpenArray AccuFill System from Thermo Fisher Scientific and run the preamplification product as described manufacturer on QuantStudio 12K Flex RT-PCR machine from Thermo Fisher Scientific, USA
Hybridization protocol
N/A
Scan protocol
N/A
Description
Raw data file: DYU32.xlxs Control 17
Data processing
The raw data was analyzed by ExpressionSuite software and Relative quantification of miRNAs expression was calculated by -ΔΔCt method, selecting U6 snRNA level as endogenous control. software performs all deltadeltaCt based fold-change calculations and performed student t-test to calculate p-value from the uploaded raw threshold cycle data . Normalized values and fold-change data are available on the series record.
Transcriptomics and proteomics approach for the identification of altered blood microRNAs and plasma proteins in Parkinson’s disease by Custom Brain Specific miRNA OpenArray Real-time PCR panel
