Is it prudent to use an RNA sample which has no RIN number, when analyzed using a Bio-analyzer (Agilent).
The profile shows a good 18S peak. However, the 28S peak is very small.(probably the reason for the absence of the RIN number)
I am attaching the picture of the profile herewith.
Can someone please look at it and tell me if it would be worthwhile pursuing this sample for hybridization for microarray?
Even if the microarray can be done and is ok, how much reproducible the data would be?
Do you have only one single sample? Then you can't say much in any case.
If you have other samples: if all profiles look like this, then they will be comparable, so you can calculate valid expression changes between conditions.
If this sample looks typical for one group but the profiles of the other group look different, then you can not sensibly compare the groups.
If this is the only "strange" sample and there are enough other "good" samples, then you can either skip this one (without loosing too much information) or hybridize it and see if the expression profile matches that of the other samples (if not: discard it).
If there are not enough other "good" samples that you must decide on the neccesity: if the samle is utmost precious then I woul use it anyway. If it is relatively simple to get another sample (hoping that it will be better) then I would go for that option.
You won't get information about the reproducibility. I don't know any study that analyzed this. If you have a bunch of samples you can estimate the reproducibility yourself (the confidence intervals [CIs] give you an estimate of the variability). When the sample is behaving strange then the CIs will be wider (but this effect will decrease with increasing sample size). Eventually you must decide if the obtained presicion is good enough for your purpose.
Do you have only one single sample? Then you can't say much in any case.
If you have other samples: if all profiles look like this, then they will be comparable, so you can calculate valid expression changes between conditions.
If this sample looks typical for one group but the profiles of the other group look different, then you can not sensibly compare the groups.
If this is the only "strange" sample and there are enough other "good" samples, then you can either skip this one (without loosing too much information) or hybridize it and see if the expression profile matches that of the other samples (if not: discard it).
If there are not enough other "good" samples that you must decide on the neccesity: if the samle is utmost precious then I woul use it anyway. If it is relatively simple to get another sample (hoping that it will be better) then I would go for that option.
You won't get information about the reproducibility. I don't know any study that analyzed this. If you have a bunch of samples you can estimate the reproducibility yourself (the confidence intervals [CIs] give you an estimate of the variability). When the sample is behaving strange then the CIs will be wider (but this effect will decrease with increasing sample size). Eventually you must decide if the obtained presicion is good enough for your purpose.
Hi Mallu. I woukd just add to Jochems excellent answer that apart from your ribosomal subunit peaks and tRNA/ 5srRNA peaks upstream from your main ribosomal species you could in my estimation/ experience have an extra peak corresponding to GITC ( derived from you RNA lysis buffer and first wash buffer if using an RNA extraction kit). What is your 260/230 ratio ? If < 1.0 this almost certainly demonstrates GITC contamination which is not advisable for array analysis. A few years back I spent the best part of 3 years purifying RNA, checking quality by nano drop and Agilent biochip and then interrogating affymetrix 3' expression arrays: when I encountered a sample that looked like that with a 260/230 ratio < 1.0 I would re purify or as suggested simply exclude. The good news is that GITC which inhibits RNA amplification and labelling prior to application to micro arrays is nearly always removed by re purification
@ Stuart, I do not have the info regarding the OD 260/230 ratio with me. Will get that
@ Burzynski, These are mammalian samples.
Please do Mallu. If you find less than 1.0 in conjunction with that 4th prominent peak in your bio analyser trace I would either exclude or re purify as duscussed
You did not mention how you isolated your sample of RNA. Did you use a method involving organic solvents or did you use a procedure using columns (e.g. Qiagen RNAeasy). The latter is vastly preferable if you will be using RNA samples for microarrays.
Presumably your sample should be total RNA; a RIN number is therefore expected. In the absence if a RIN number we would certainly discard the sample.
If the OD 260/230 ratio < 1 we would not use a sample for microarrays. Re-purification is certainly possible. Best solution is to get further samples as already suggested, and to use a column-based isolation kit.
Mallu - What is the organism that you are working with? Some organisms like Drosophila have non-typical bioanalyzer pattern. For example, in Drosophila, its 28S rRNA is split into 2 fragments, and they co-migrate with the 18S rRNA. So yours might be normal if you are working with such a species.
Hi
For microarray you will need a minimum concentration of RNA as 50ng/ul and a RIN value of 7 and good 260/280 ratio. I think you RNA conc is around 6.6ng/ul if I am right. Would recommend to isolate RNA using Qiagen or Ambion kits
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA