I am using 74904 to extract RNA from wheat seed and I have tried both of the lysis buffer. As it is mentioned it protocol, buffer RLT is the lysis buffer of choice but it may cause solidification of some samples which in this case, buffer RLC should be used. I have tried both and both of them caused solidification. What is your recommendation?
Hi Somayeh,
This thread has some suggestions for your question. I hope that it may solve your problem,
https://www.researchgate.net/post/Does_anyone_know_how_to_extract_RNA_from_samples_with_high_starch_content_wheat_kernels_using_RNeasy_Plant_Minikit
You should use liquid nitrogen twice.
Also, I recommend that you use extraction kits with a collum filter.
Hi, you can also check this other kit from Qiagen that is supposed to work with seeds:
https://www.qiagen.com/us/products/diagnostics-and-clinical-research/sample-processing/rneasy-powerplant-kit/#orderinginformation
I have not tried myself with seeds, but maybe you can try.
Abozar Ghorbani , Thank you for your consideration. The kit contains column filter tube. Would you please clarify what do you mean by using liquid nitrogen twice?
Diana Duarte-Delgado Thank you for your response, Qiagen specialist also recommended to use PVP.
S M Zia Hasan Thank you for your response.
Your crushed tissue should show white color, It is important you don't let a tissue be melting before well crushing and you add lysis buffer before melting.
-Reduce the amount of starting material and/or increase the volume of lysis buffer (0.5 to 1 additional volume).
-Cool the lysis buffer and vortex it before usage.
-Keep all stuff on ice (even your pestle and mortar or whatever you are using as your container of the tissue sample).
-RNA should dissolve faster than starch otherwise viscosity will increase; thus, work so fast! As Abozar Ghorbani suggested if during the homogenization the plant tissue started melting, add more liquid nitrogen.
By the way, you can try TRIzol reagent, or even phenol:chloroform-based methods. According to my experience, in this kind of methods, there is more flexibility and you can manipulate the condition in favor of your issues (e.g. adding Dithiothreitol (DTT) to decrease the probable oxidation for tissues with high polysaccharides and secondary metabolites), and PVP for tissues with high polyphenolic pollution) to get a high-quality RNA.
Maybe this paper helps you:
Article A method for extracting high-quality total RNA from plant ri...
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