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j123's Content

There have been 12 items by j123 (Search limited from 25-October 19)


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#61663 If we somehow downregulate pri-miRNA, mature miRNA will also be downregulated?

Posted by j123 on 08 March 2010 - 09:10 AM in siRNA, microRNA and RNAi

I downregulated one gene mRNA level using siRNA. Then interesting thing is the miRNA, which is located in this gene, expression level was also downregulated. But my previous experiments showed that the miRNA and the host gene use different promoter. So my explaination is that the siRNA downregulated miRNA by downregulating pri-miRNA. Does it make any sense? Please help me with this. I am so confused. :)



#58381 Optimizing transfection of miRNA

Posted by j123 on 10 February 2010 - 08:53 AM in siRNA, microRNA and RNAi

Try NeoFX transfection reagents from AB. It works very well for me. It also depends on different cells. Good luck!


Hello, I need some advice because I am stuck....

I am trying to knockdown c-Myc and/or RAS using miRNA, let-7.

This is basic protocol I used.

- 12-well plate
- 150,000 cells per well. (HeLa, A549, Panc-1, MB231)
- Total transfection volume: 1250 microL (1000 microL of growth and 250 microL of diluted medium)
- 80nM of miRNA per well
- Tried 1, 2, and 4 microL of Lipofectamine 2000
- 2 days transfection

Need advice on what I should try next.

Thank you very much




#56962 pre-miRNA existence

Posted by j123 on 28 January 2010 - 01:49 PM in siRNA, microRNA and RNAi

Thanks a lot. It really helps.


I ordered the MGB probe with FAM Dye, since it binds to the loop part of the pre-miRNA. It is a hydrolysis probe which will give out signal at the elongation stage of the PCR.
Of course you will need sense and antisense primers binding to the 5' and 3' end of the pre-miRNA sequence, respectively. Check the guideline for primer design. They have relative strict rules for the sequence and Tm for both primers and probe.
Not familiar with TAMRA probe.

I check AB website, there are two kinds of probes :
* TaqManŽ MGB Probes
* TaqManŽ TAMRA™ Probes

Could you please tell me which one I should order? Thanks again for your kind help.





Yes, they can make customerized Taqman probe for you. It costs about $230, and a week's wait. Use their website for ordering.

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz




#56950 pre-miRNA existence

Posted by j123 on 28 January 2010 - 12:59 PM in siRNA, microRNA and RNAi

I check AB website, there are two kinds of probes :
* TaqManŽ MGB Probes
* TaqManŽ TAMRA™ Probes

Could you please tell me which one I should order? Thanks again for your kind help.





Yes, they can make customerized Taqman probe for you. It costs about $230, and a week's wait. Use their website for ordering.

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz




#56949 pre-miRNA existence

Posted by j123 on 28 January 2010 - 12:55 PM in siRNA, microRNA and RNAi

Thanks a lot for the information. I really appreciate it.



#56785 pre-miRNA existence

Posted by j123 on 27 January 2010 - 10:47 AM in siRNA, microRNA and RNAi

Hi, Fizban, could you please send me the link? Thank you very much for the helpful discussion.


Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


Here in italy custom probes design and validation is available. you have just to wait for validation of your probes but shouldn't take long.
If it works in italy....it works everywhere!
:D sad but almost always true
bye Fiz




#56768 pre-miRNA existence

Posted by j123 on 27 January 2010 - 07:12 AM in siRNA, microRNA and RNAi

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.


I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.


You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz




#56713 pre-miRNA existence

Posted by j123 on 26 January 2010 - 03:31 PM in siRNA, microRNA and RNAi

I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.



#56073 miRNA promoter

Posted by j123 on 21 January 2010 - 08:37 AM in siRNA, microRNA and RNAi

That's a very good idea. I am trying to treat cells to check the miRNA expression. Thanks a lot.




First you can do some bioinformatics analysis to see if you can find if the sequence upstream of the miRNA has promoter features such as CpG island, TATA box, etc.

If the miRNA uses the promoter of its host gene, they may be coexpressed. So check their expression in different cell lines may help. Can the transcription of the host gene be manipulated by some treatment? if so, treat your cells to see if the expression of the miRNA also changes.




#55956 miRNA promoter

Posted by j123 on 20 January 2010 - 12:45 PM in siRNA, microRNA and RNAi

Thanks for the response. I mean intragenic, not intergenic. It seems some intronic miRNAs (intragenic) use unique promtors, which are different from the host promoters. What kind of experiments I can do to test if they share the same promoter? I detected endogenous miRNA expression in cancer cell lines by RT-PCR. Some cell lines the expression level is very high. Thanks again for kind help.




Intergenic is different to intronic. But anyway, have you checked expression of any endogenous miRNA in your cell line? Some cancers and perhaps cancer cell lines have mutations in the miRNA biogenesis machinery.

As to Jon's questions, perhaps the Drosha processing isn't very efficient and so not all of the transcript is processed to pre-miRNA, leaving some of the host mRNA intact? I suppose though that if the lariat is long enough and has the correct secondary structure and/or Drosha recognition sequence then it could be precessed. As far as I know (and please correct me) the recognition sequence for Drosha hasn't been determined? If we knew that, it might be a lot easier to understand!




#55856 miRNA promoter

Posted by j123 on 19 January 2010 - 01:11 PM in siRNA, microRNA and RNAi

Thanks for your reply. One paper says that 26% of intragenic miRNAs may be transcribed from their own unique promoters. I used Tet-on Advanced system to express the whole host gene, and checked the miRNA expession by RT-PCR. I didn't see the increase of miRNA although the host gene expression dramastically increased. Thus, I think the miRNA I am interested in and the host gene come from different transcripts. But I am not sure if they share the same promoter. Any suggestions? Thanks again for your kind discussion.




I am pretty new to this field. Does anybody know how to find the promoter region of an intronic miRNA? How to distinguish the intronic miRNA promoter with its host gene promoter? Many thanks.


I don't think the intronic miRNA has an independent promoter, but rather that the host gene promoter causes transcription of the pre-mRNA containing the intronic miRNA and then the intronic miRNA is cleaved from the intron by Drosha. I am curious whether this is thought to happen after splicing, so that the miRNA is cleaved from the intron lariat. I don't see how it could happen prior to lariat formation without disrupting the pre-mRNA by cleaving the pre-mRNA into two pieces.

Please chime in!




#55850 miRNA promoter

Posted by j123 on 19 January 2010 - 12:01 PM in siRNA, microRNA and RNAi

I am pretty new to this field. Does anybody know how to find the promoter region of an intronic miRNA? How to distinguish the intronic miRNA promoter with its host gene promoter? Many thanks.




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