2 replies to this topic

[marek82313]

Hi everybody,

I'm new here. I started few days ago extracting cytoplasmic RNA using TRIzol from Invitrogen. Since I need to remove genomic DNA contamination prior reverse transcription of the RNA, I've performed a DNase I digestion of my samples (using RQ RNase Free DNase from Promega). The samples were treated as protocol and the RNA was precipitated with iropropanol, washed with 75 % ethanol (twice) and dissolved in DEPC water.
Here is what I got after running my samples in a 1.5% agarose gel; left DNase treated samples, right samples prior DNase treatment (excetp the first samples to the right that are 0.2 ul of DNase treated RNA). Are the RNA degraded by the treatment (maybe because of RNase contamination)? or are there too much salts (I've read in the DNase usage information sheet that the 10X DNase buffer and stop solution contain salts that can cause aberrant migration on gel).

Does anybody know any trick for RNA purification ? I was wondering if I can digest directly in the aqueous phase after chloroform addition and than recover the RNA adding again 1 ml of TRIzol and following the original protocol (chloroform, isopropanol and so on).

Thank you in advance

Sorry for my very bad english.

Here is the picture of my gel

[bob1]

marek82313, on Mar 17 2009, 12:08 PM, said:

Hi everybody,

I'm new here. I started few days ago extracting cytoplasmic RNA using TRIzol from Invitrogen. Since I need to remove genomic DNA contamination prior reverse transcription of the RNA, I've performed a DNase I digestion of my samples (using RQ RNase Free DNase from Promega). The samples were treated as protocol and the RNA was precipitated with iropropanol, washed with 75 % ethanol (twice) and dissolved in DEPC water.
Here is what I got after running my samples in a 1.5% agarose gel; left DNase treated samples, right samples prior DNase treatment (excetp the first samples to the right that are 0.2 ul of DNase treated RNA). Are the RNA degraded by the treatment (maybe because of RNase contamination)? or are there too much salts (I've read in the DNase usage information sheet that the 10X DNase buffer and stop solution contain salts that can cause aberrant migration on gel).

Does anybody know any trick for RNA purification ? I was wondering if I can digest directly in the aqueous phase after chloroform addition and than recover the RNA adding again 1 ml of TRIzol and following the original protocol (chloroform, isopropanol and so on).

Thank you in advance

Sorry for my very bad english.

Here is the picture of my gel
gel.jpg

The RNA seems to be fine, DNases and RNases often have some non-specific activity in that they will work on both DNA and RNA to a limited extent.  Were both the DNase and DNase free samples treated in exactly the same fashion  (i.e. only difference is the DNase treatment)?  If not, then it may be because you have extracted the RNA one more time than the untreated samples.

[marek82313]

bob1, on Mar 18 2009, 12:37 AM, said:

marek82313, on Mar 17 2009, 12:08 PM, said:

Hi everybody,

I'm new here. I started few days ago extracting cytoplasmic RNA using TRIzol from Invitrogen. Since I need to remove genomic DNA contamination prior reverse transcription of the RNA, I've performed a DNase I digestion of my samples (using RQ RNase Free DNase from Promega). The samples were treated as protocol and the RNA was precipitated with iropropanol, washed with 75 % ethanol (twice) and dissolved in DEPC water.
Here is what I got after running my samples in a 1.5% agarose gel; left DNase treated samples, right samples prior DNase treatment (excetp the first samples to the right that are 0.2 ul of DNase treated RNA). Are the RNA degraded by the treatment (maybe because of RNase contamination)? or are there too much salts (I've read in the DNase usage information sheet that the 10X DNase buffer and stop solution contain salts that can cause aberrant migration on gel).

Does anybody know any trick for RNA purification ? I was wondering if I can digest directly in the aqueous phase after chloroform addition and than recover the RNA adding again 1 ml of TRIzol and following the original protocol (chloroform, isopropanol and so on).

Thank you in advance

Sorry for my very bad english.

Here is the picture of my gel
gel.jpg

The RNA seems to be fine, DNases and RNases often have some non-specific activity in that they will work on both DNA and RNA to a limited extent.  Were both the DNase and DNase free samples treated in exactly the same fashion  (i.e. only difference is the DNase treatment)?  If not, then it may be because you have extracted the RNA one more time than the untreated samples.

Hi bob1,

thank you for your help,
the samples were treated in the same fashion, going more in detail, the I've extracted the RNA, saved few microliters (the "not-treated RNAs" on the right), DNase treated the remaining volume of RNA solution, re-precipitated them through isopropanol-ethanol precipitation. The DNase-treated RNAs were dissolved in DEPC-water. The only difference I can think about is the volume of water I used to dissolve the RNA, during the first precipitation I dissolved the RNA in 40 microliters, while during the second precipitation 22-28 microliter (according to the size of the pellet) were used.

I was puzzled (and worried ) about the appearance of the RNAs after treatment, they look smeared and the 28S and 18S bands appear so less discrete and "bulky" comparing to the ones in the RNAs before the treatment...