RNA extraction - didnt get ratio 2:1 ( 28s rRNA : 18 s rRNA) - RNA extraction from Tilapia (Mar/11/2007 )
I did my RNA extraction from the gills of Tilapia using RNeasy Kit by Qiagen. I ran the my samples in 1.0 % native agarose gel and I used 1x TAE buffer (DEPC-treated ) as my running buffer. I used 2x RNA loading buffer ( bought from Fermentas) and I incubated an equal volume of RNA loading buffer with my samples at 70 degree celcius for 10 minutes to denature my RNA sample (as mentioned in protocol). I run the gel electrophoresis for 80 V for 1.5 hours.
Well, the problem is i got 2 bands but one was not clear compared to the other one. i got vey blur 28s rRNA and a very good and sharp 18s rRNA ( the ratio was something like 1: 2). This was different from the standard whereby the 28s rRNA should be twice than the 18s rRNA.
I ran my samples together with the Promega RNA marker and the sizes were approximately 3638bp for the 28s rRNA and inbetween 1385 bp and 1908 bp.
My question is how to improve my RNA extraction so that I will get a good 28r RNA and 18 r RNA with the ratio of 2:1 ?
Please help me ?
by those kits you couldnt get the actual ratios and you have to modify existing protocols for best resault on your organism,
but it is not really important if the ratio is not correct for normal lab activities.
I did RNA extraction from tilapia again and this time I got 2 sharp bands and yet I didnt get 2: 1 ratio. I am going to run Reverse Transcriptase PCR ( RT-PCR). Does these results of mine will effect my RT-PCR ?