LncRNA METING TEMPRATURE?

Fatma Abdel Wahab @Fatma_Abdel_Wahab

12 December 2017 1 3K Report

Hi all I use total RNA EXTRACTION KIT ( QIAGEN).

THEN I USE SYBR GREEN BASE RT PCR to antiquate HOTAIR LncRNA

What is the accepted change in Tm between run of my product

Binayak Raj Pandey

First you need to go to RNA central database to know the sequence of your LncRNA

http://rnacentral.org/expert-database/lncrnadb

Then to calculate Tm Two standard approximation calculations are used.

For sequences less than 14 nucleotides the formula is:

Tm= (wA+xT) * 2 + (yG+zC) * 4

where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

For sequences longer than 13 nucleotides, the equation used is

Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

With SYBR Green PCR Kit, HotStarTaq DNA Polymerase are provided with 15 min activation at 95ºC and you can set-up qPCR reactions at room temperature.

You can also download handbook for RT2 lncRNA qPCR Assay :

https://www.qiagen.com/np/resources/resourcedetail?id=ba35e739-328f-4f9e-8ade-1c98065abda0&lang=en

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