Placental RNA extraction with qiagen rneasy mini plus kit
Posted 05 March 2013 - 05:59 AM
I am trying to extracted RNA from human placenta using RNeasy mini plus kit. The protocol followed was as mentioned by the user manual and when sufficient yield was not obtained, some variation in protocol was made. However, I am getting continousely very low yield. The initial tissue taken was 30 mg from which I obtained 100ng/ul concentration (nanodrop reading), RNA elution was done by 15ul Rnase free water and A260/280 was 2.01.
I want following suggestion:
1. What should I do to obtain more yield?
2. How much yield will you get normally from placental tissue?
Posted 05 March 2013 - 11:42 AM
Posted 05 March 2013 - 08:26 PM
Yes, i have seen the RNA on gel. It shows 28S and 18S bands and a smear starting from just above the 28S till below of 18S. The quantity on gel also looks very less.
Posted 06 March 2013 - 01:06 PM
As to the yield issues - have you tried a Trizol (phenol:chloroform) extraction or another kit? Typically it is not a good idea to modify the kit procedures, they have been optimised for the kit. Perhaps using 30 mg is too much for one of the steps (probably the digestion/disruption step), have you tried to use less tissue in an extraction?
Posted 06 March 2013 - 08:23 PM
My concern is the sample amount which I get is very less. With trizol method I am losing a lot of tissue which I cant afford, plus the purity is not the same how i get with kit.
I have tried using low amount of tissue (5,10 and 20 mg) also for the kit method, but the results i get on gel are the same. Following are the experiments i did: Sample.doc 39.5K 27 downloads
Edited by sheon, 06 March 2013 - 08:30 PM.
Posted 06 March 2013 - 11:11 PM
Posted 06 March 2013 - 11:40 PM
I wanted to know how much is normally the RNA YIELD from placental tissue.
Posted 07 March 2013 - 12:02 PM
Posted 10 March 2013 - 12:14 AM
I tried combining trizol method and kit method together. I am getting a yield of around 800ng/ul from 30mg tissue with purity A280/260 of 2.01.
Posted 10 March 2013 - 11:42 AM