3:53 Both forward and reverse primer are drawn at the same strand of 5' adapter and universal RT primer. Primer can not hybridize like this because primer is the same direction against a template.
Please tell me any one that HOW many types of microRNAs profile are detected by Taqman microRNA assay kit.?Is this kit is only used for detection of a single microRNAs profile or for multiple types of microRNAs profiles.
Due to how the assays are designed, in general we do not see any off target amplification. In fact, the majority of assays have < 5% cross reactivity with closely related sequences. It is well understood within the miRNA community that designing assays for miRNAs is challenging due to their short length ( 0.98).
Hey Marcus, we need a little more information to answer this for you. If you fill out the form in the link below, someone will follow up with you ASAP: www.thermofisher.com/us/en/home/technical-resources/contact-us/ask-a-question1.html
Thank you for reaching out. As there are multiple techniques for quantifying microRNA’s, the right technique for you will depend upon what specific information you need to obtain. For example, if you are studying the expression of microRNA-31 across different tissue samples to compare the changes in microRNA-31 in response to a specific condition or treatment, we would recommend the Comparative Ct method for data analysis along with the use of our traditional TaqMan MicroRNA Assays. However, if you were looking to profile the abundance of a panel of microRNA species including microRNA-31, we would recommend the use of our TaqMan Advanced miRNA Assays. You can reach us at techsupport@thermofisher.com if you have more questions.
Hi Ameet, Choosing the appropriate endogenous control will depend on several factors. As each qPCR assay is different, it is best to tailor the endogenous control to the specific target gene(s) of interest. The ideal endogenous control will be present in similar abundance to your target gene(s), have similar amplification efficiency, and most importantly be stably expressed across all of your samples and conditions. You may need to experimentally validate one or several candidate controls to see which is the best fit for your experiment. For microRNA studies, we offer a number of candidate endogenous control assays including assays for the most popular microRNA controls (i.e. U6 snRNA). The selection of these assays is discussed in this Application Note: tools.thermofisher.com/content/sfs/brochures/cms_044972.pdf Please let us know if you have any other questions.
Hi M, Thanks for your question. If you are looking at only a few TaqMan miRNA assays, we recommend using the TaqMan miRNA assays. The TaqMan Advanced miRNA system is more for researchers looking to detect more than 10 miRNA targets. If you are looking at isomirs that have similar nucleotides on the 5’ end, we recommend the TaqMan Advanced miRNA system. The TaqMan miRNA system isn’t able to differentiate at the 5’ end as well when comparing similar miRNAs. If you are looking to use U6 as an endogenous control, we recommend using the TaqMan miRNA assay as the Advanced miRNA system requires a phosphate on the 5’ end of the miRNA, which snoRNA/snRNAs do not have. For additional technical support, please contact us at thermofisher.com/askaquestion. Thank you!
i am working on salivary microRNA 31. plz tell me regarding universal master mix, which master mix should i use taqman universal master mix with UNG or without UNG?
Hey Fatima, Thanks for reaching out to us with this question. If you can go to www.thermofisher.com/askaquestion and give us a more detailed description of the issue you're having, we can better help you resolve it. Thank you!
is the control prime/probe supposed to be added along with the specific primer/probe in case of PCR ( both reverse transcription and real time) ? I m.working with taqman mir124 primers and probes and using U6 snRNA as control.
Hi Uzma, With TaqMan MicroRNA Assays, our protocol allows for multiple stem-loop RT primers to be pooled in the same reaction to generate cDNA. In the case of two assays, we recommend keeping the same total volume of 5X RT primers in the RT reaction and reducing the amount of RT primer added to half the recommended amount for each assay (1.5uL control + 1.5uL target). By default, our pre-designed TaqMan MicroRNA Assays are labeled with the FAM reporter dye. For this reason, we do not recommend adding both assays to the same qPCR reaction. Hope that helps but if you need more answers, you can always reach us at techsupport@thermofisher.com for more assistance.
한국어 지원이라니 짱이다...!
3:53 Both forward and reverse primer are drawn at the same strand of 5' adapter and universal RT primer. Primer can not hybridize like this because primer is the same direction against a template.
I isolate total RNA from plasma can I use TaqMan MicroRNA Assays to synthesis cDNA to perform QRT-PCR to evaluate mRNA level of protein ?
Please tell me any one that HOW many types of microRNAs profile are detected by Taqman microRNA assay kit.?Is this kit is only used for detection of a single microRNAs profile or for multiple types of microRNAs profiles.
I need micro RNA 122,and 121 kit.
Which type of TaqaMan kit i used.?
Please send me their price of selected kit.
Hello, using MicroRNA transcription kit and Taq Man microRNA assay... is possible amplify off-targets on RT-qPCR?
Due to how the assays are designed, in general we do not see any off target amplification. In fact, the majority of assays have < 5% cross reactivity with closely related sequences. It is well understood within the miRNA community that designing assays for miRNAs is challenging due to their short length ( 0.98).
For which one assay exogenous control is required? For taqman miRNA assays or taqman advanced miRNA assay or both
If I want to creat cDNA library from all miRNA available in in plant tissue then which will be the best way to proceed plz reply
For the advanced miRNA assays, how do you only measure the miRNAs that you are interested in if all of the miRNAs in the sample are amplified?
Hey Marcus, we need a little more information to answer this for you. If you fill out the form in the link below, someone will follow up with you ASAP:
www.thermofisher.com/us/en/home/technical-resources/contact-us/ask-a-question1.html
2021 this is very relevant
which technique should i use for quantifying microRNA- 31
Thank you for reaching out. As there are multiple techniques for quantifying microRNA’s, the right technique for you will depend upon what specific information you need to obtain. For example, if you are studying the expression of microRNA-31 across different tissue samples to compare the changes in microRNA-31 in response to a specific condition or treatment, we would recommend the Comparative Ct method for data analysis along with the use of our traditional TaqMan MicroRNA Assays. However, if you were looking to profile the abundance of a panel of microRNA species including microRNA-31, we would recommend the use of our TaqMan Advanced miRNA Assays. You can reach us at techsupport@thermofisher.com if you have more questions.
thank you for your response.. i am studying on the expression of microRNA-31 in oral cancer patients by means of saliva.
i have query regarding which control will be more stable in saliva?
Hi Ameet,
Choosing the appropriate endogenous control will depend on several factors. As each qPCR assay is different, it is best to tailor the endogenous control to the specific target gene(s) of interest. The ideal endogenous control will be present in similar abundance to your target gene(s), have similar amplification efficiency, and most importantly be stably expressed across all of your samples and conditions. You may need to experimentally validate one or several candidate controls to see which is the best fit for your experiment.
For microRNA studies, we offer a number of candidate endogenous control assays including assays for the most popular microRNA controls (i.e. U6 snRNA). The selection of these assays is discussed in this Application Note:
tools.thermofisher.com/content/sfs/brochures/cms_044972.pdf
Please let us know if you have any other questions.
what are the advantages and disadvantages of taqMan microRNA assay?
Hi M,
Thanks for your question. If you are looking at only a few TaqMan miRNA assays, we recommend using the TaqMan miRNA assays. The TaqMan Advanced miRNA system is more for researchers looking to detect more than 10 miRNA targets.
If you are looking at isomirs that have similar nucleotides on the 5’ end, we recommend the TaqMan Advanced miRNA system. The TaqMan miRNA system isn’t able to differentiate at the 5’ end as well when comparing similar miRNAs.
If you are looking to use U6 as an endogenous control, we recommend using the TaqMan miRNA assay as the Advanced miRNA system requires a phosphate on the 5’ end of the miRNA, which snoRNA/snRNAs do not have.
For additional technical support, please contact us at thermofisher.com/askaquestion. Thank you!
i am using taqman microrna assay.. is it necessary to use reverse transcription kit? as i studied stem loop primer is present in taqman microrna assay
i am working on salivary microRNA 31. plz tell me regarding universal master mix, which master mix should i use taqman universal master mix with UNG or without UNG?
can you please tell me how did you collected salivary samples and how did you extracted mirna from it?
How I can determine the SNPs of miRNA? I don't understand how they isolate DNA then perform rflp-PCR or real time-pcr can anyone help me please
How to use the U6 to normalize? We should by a probe to U6???
Hey Fatima,
Thanks for reaching out to us with this question. If you can go to www.thermofisher.com/askaquestion and give us a more detailed description of the issue you're having, we can better help you resolve it. Thank you!
is the control prime/probe supposed to be added along with the specific primer/probe in case of PCR ( both reverse transcription and real time) ? I m.working with taqman mir124 primers and probes and using U6 snRNA as control.
Hi Uzma, With TaqMan MicroRNA Assays, our protocol allows for multiple stem-loop RT primers to be pooled in the same reaction to generate cDNA. In the case of two assays, we recommend keeping the same total volume of 5X RT primers in the RT reaction and reducing the amount of RT primer added to half the recommended amount for each assay (1.5uL control + 1.5uL target). By default, our pre-designed TaqMan MicroRNA Assays are labeled with the FAM reporter dye. For this reason, we do not recommend adding both assays to the same qPCR reaction. Hope that helps but if you need more answers, you can always reach us at techsupport@thermofisher.com for more assistance.