Page 37 - IBRO_RNA School_Abstract Book

Protocols for virtual lab sessions

Virtual Lab session: miRNA profiling in human serum

Prachi Singh

CSIR-Institute of Genomics and Integrative Biology,

Mathura Road, Delhi, India

Collection of serum from blood:

Collect blood samples from the patient in uncoated vacutainers (red co-

loured cap). Keep the samples at room temperature for 20 to 30 minutes fol-
lowed by centrifugation at 1000g-2000g for 10 minutes at 4°C for separation
of serum from blood.

After the centrifugation carefully transfer the serum (supernatant, straw co-
loured) to a separate tube. Add 3 volumes of Trizol-LS to the serum samples

that is to be used for RNA isolation. Store the samples at -20°C.

RNA isolation:

RNA from the sample is isolated using the Trizol reagent based on the

phase separation principle. Upon homogenizing sample in Trizol,
Chloroform is added to facilitate phase separation and then RNA is
precipitated using Iso-propanol. Precipitation can be enhanced using the
reagents like Glyco-gen.

cDNA synthesis:

cDNA synthesis for microRNA detection is a 3-step procedure when it is
done manually.

PolyA tailing

Due to small size of microRNA, primer pair (forward and reverse) cannot

bind to it and it needs an adaptor for binding of either of the primer so that it
can be detected using qRT-PCR. With the help of PolyA polymerase, RNA is
Poly adenylated by incubating at 37°C/30’ and 85°C/10’. Poly A tail will fur-

ther help in binding of reverse primer adaptor on the microRNA.