RNA extraction from hepatopancreas of shrimp,

We use modified procedure of Solution D method (Guanidinium thiocyanate-phenol-chloroform extraction method) to extract total RNA from hepatopancreas of shrimp. Since, it has guanidinium thiocayanate, a strong chaotropic agent to prevent activity of RNase and DNase enzymes by denaturing them effectively. We always get good quality and quantity of Total RNA from fresh tissues of hepatopancreas than stored tissue samples. Degradative enzymes such as RNase and DNase rich in organs like pancreas from mammals and hepatopancreas from arthropods. Liquid nitrogen is the good choice for lysate preparation.

Procedure

For 100 mg of hepatopancreas tissue, add enough volume of liquid nitrogen, pulverize the tissue and transfer to a DEPC-treated tube containing 1 ml of solution D.

Allow the sample to thaw and add 0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of phenol, chloroform: isoamyl alcohol (24:1). Vortex the homogenate vigorously and incubate it on ice for 15 minutes.

Centrifuge at 12,000g for 20 minutes at 4°C and carefully transfer upper aqueous phase (80%) to a fresh centrifuge tube.

Add equal volume of isopropanol, mix well and incubate at -20°C for 30 minutes and pellet the total RNA by centrifugation at 12,000g for 15 minutes at 4°C.

Discard the supernatant and wash the pellet with 70% ethanol by centrifugation at 12,000g for 15 minutes at 4°C.

Briefly air-dry the pellet and re-suspend the total RNA in appropriate amount of DEPC-treated water and store at -70°C. Since, mRNA has a short half-life period, convert it to mRNA:cDNA hybrid ASAP.

I hope this may be useful to your work!

Yes, I can definitely support Aravindans answer. Use GTC method and phenol chloroform isoamyl. It is dirty (be careful!) but cheap and best way ever. It might take some tricks and time to get best quality but if yo7 need help let me know. Important: don’t use denatured isoprop. you can never be sure if there aren’t any substances used for the dental ration which might influence your isolation. Eg Toluol can be one of them.

Let me know if you want more information on tips and trickS I have always used. Good luck

Hi, we injected RNA-later dierctly into the pancreas to protect from RNAses. We also used MACE-Seq instead of regular RNA-seq as the method is less prone to RNA degradation (https://genxpro.net/sequencing/transcriptome/mace-massive-analysis-of-cdna-ends/).

Teresa Otto, Do you mind to send me a detailed protocol? it will be nice for me..I am still struggling with such procedure and my RINs do not improve at the moment!@Teresa otto

no, I don’t mind At all. Just send me your email address in a private message and I will send it to you on the weekend. Sorry, but I am just standing in front of an internal audit at work and I have to do a lot of work -still. :) so i hope you dont mind waiting for it until the weekend.