miScript SYBR Green PCR Kit (2000)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Follow manufacturer’s instructions

Protocol tips
Follow manufacturer’s instructions

Publication protocol

Real-time PCR for Detection of miRNA
Real time PCR for miRNAs was performed using miScript SYBR Green PCR Kit and miScript Primer Assay according to manufacturer's instructions (Qiagen). Prepare a master mix containing 2x QuantiTect SYBR Green PCR Master Mix, 10x miScript Universal Primer, 10x miScript Primer Assay, and RNase-free water. Prepare a master mix for a 20 μl volume reaction.
The Primer Assay is specific to the miRNA of interest. To reconstitute 10x miScript Primer Assay, centrifuge the vial briefly, and add 550 μl TE buffer, pH 8.0. Vortex the vial briefly to mix, aliquot primers to smaller volumes, and store at -20 °C. Two primers are required: primers for the target gene and the reference gene. RNU6B is usedas the reference gene.
Dilute the cDNA 40x and store extra aliquots at -20 °C.
cDNA serves as the template for the PCR. Use 2 μl of 40x diluted cDNA and dispense to the 20 μl light cycler capillaries (Roche).
Add 18 μl of the master mix to each capillary, and centrifuge using a capillary adapter.
Place the capillaries in a capillary based Real-Time cycler, such as LightCycler 3.5 Real-Time PCR System with a 32-capillary carousel format.
Run the PCR cycling program as follows: To activate HotStarTaq Polymerase that is in the 2x QuantiTect SYBR Green PCR Master Mix, pre-incubate at 95 °C for 15 minutes. Followed by 50 cycles of: Denaturation, 15 s, 94 °C; Annealing, 30 s, 55 °C; Extension, 30 s, 70 °C.
Select a sample to be the calibrator, and set its normalized target amount to 1. Compare the relative expression of the miRNA in all the other samples to the calibrator.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing PCR Quantitative real-time PCR - Mammalian DNA using miScript SYBR Green PCR Kit (2000) from Qiagen.

Manufacturer protocol

Download the product protocol from Qiagen for miScript SYBR Green PCR Kit (2000) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing PCR Quantitative real-time PCR - Mammalian DNA using miScript SYBR Green PCR Kit (2000) from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.