General Lab techinique - RNA isolation (Jun/15/2006 )
The 260/280 ratio of my RNA samples always comes around 1.6-1.8 . I need high quality RNA samples for cDNA synthsis followed by gene expression assay by RT-PCR. Can any one suggest me how to to obtain high quality RNA ? and How to quantify cDNA?
I use Trizol from Invitrogen and I get a ratio around 1.8 as well. Honestly I don't think it's bad and I never have problems when I do real-time PCR afterwards.
When I do the real-time, I never quantify the cDNA because of two reasons:
- you quantify the RNA and you are supposed to get the same reverse transcription efficiency for all your samples if you use the same oligos
- you always use a control for quantification by real-time, and that will ´give you an indication of how much your starting material was.
hi devi Sivanesan! A ratio less than 1.7 means there is probably a contaminant in the solution, typically either protein or phenol.
phonol contamination with in RNA preparations decreases the 260/280 ratio. check for phenolic contamination. phenols absorb strongly at 270 nm.
if it is phenolic contamination, then add chloroform to the RNA preparation and perform phase separation, ethanol precipitation, pelleting and redissolving in DEPC tr. TDW.
if you think it is protein contamination, try adding little excess of chloroform during TRIZOL phase separation, increase centrifugation speed/ time and also try to take only uppermost part of the solution avoiding most of the upper aqueos phase (you have to sacrifice some amount of upper aqueous phase. this is because as you move to the interphase, DNA concentration increases and also protein adn phenols might also be present there as contaminants(due to improper phase separation)).
hope this will help you
hi 'devi Sivanesan'
i t took me around 3 years in extracting total cellular RNA from certain human tissue like bladder tissue and also extracting viral Rna from body fluids using trizole reagent although trizole is a reputable method world wide ,i faced alot of problems concerning the optimization of the methods.
below are my tips for getting a high yield and very pure Rna prep.:
1.when you use tissues perform homogenization efficiently and on small amount of tissue to give the chance for trizol to extract the proteins aand to increase the surface area for reaction .
2. after phase separation you can do extra step with chloroform-isoamyl only to get rid of excess phenol if present
3. be cautious not to aspirate near the interface
4.precipitate with ice cold isopropanole.
if a problem still i can send u my optimized protocole my mail is firstname.lastname@example.org