Now i´m working in RNA isolation of the half part or all the maxilar palate mucosa of BALB/c mouse, with the aim to measure the gene expression of different cytokines with RT-qPCR. But my problem is with the obtention of a good quantity of RNA. Now i'm using a protocol that use Trizol reagent and alcohol precipitation. Any tip or protocol to obtain more RNA?
Thank you!!
Hello Monasterio
I am working on mammalian and birds viruses and often have to isolate viral genome from body fluids and tissues. I found best the Trizol and alcohol precipitation protocol. But an other important thing you must add is to use DEPC-treated tips, tubes to ensure inactivation of RNases to enhance your RNA yield
Good Luck
Actually we use RNAsa free materials, so maybe i think the problem is in the size of the tissue sample (very little) and the homogenizate process. Wich method of homogenization do you prefer? Automatic or manual? Now i'm using glass homogenizer for manual process.
Hi Gustavo,
I will answer to you in English because this can help to someone else... To extract total RNA from murine gingival tissues, we also use Trizol and alcohol precipitation protocol with good results. What method are you using to disrupt the tissue? I think that is the key. Let me know so I can help you more
Best
Hi! now i'm using glass tissue homogenizer, with Trizol in the process.
Hi,
With glass homogenizers you need to be sure that the tissue disintegrates really well which is hard to tell because of the size of the sample. My concern is maybe you are not homogenizing the sample enough and you are loosing some part of it.
The way that we are doing the harvest and RNA extraction is: First, we harvest the gingiva, we snap freeze it in dry-ice and then we store it at -80 till we process it. Then we use an automatic tissue homoogenizer (gentleMac Dissociator from Miltenyi Biotec) with Trizol on the tubes to disrupt the tissue. Finally we extract RNA following Trizol protocol
- Badges
- Science method