I did some comparative analysis using our own qPCR, RNA-seq, and nanostring data. Looks like qPCR is still the best. Of course, it is a challenge to analyze several hundred genes using qPCR. Typically, we still need to validate nanostring data using an independent approach such as qPCR.
【在 l***s 的大作中提到】 : I did some comparative analysis using our own qPCR, RNA-seq, and nanostring : data. Looks like qPCR is still the best. Of course, it is a challenge to : analyze several hundred genes using qPCR. Typically, we still need to : validate nanostring data using an independent approach such as qPCR.
We often use qPCR to detect 30% change (with duplicated reactions). qPCR is most sensitive because of the signal ampplification process. We sometimes use qPCR for about 100 cells with good sensitivity.
【在 l***s 的大作中提到】 : We often use qPCR to detect 30% change (with duplicated reactions). qPCR is : most sensitive because of the signal ampplification process. We sometimes : use qPCR for about 100 cells with good sensitivity. : : cycle
h*a
59 楼
看看去
k*n
60 楼
second this
is
【在 l***s 的大作中提到】 : We often use qPCR to detect 30% change (with duplicated reactions). qPCR is : most sensitive because of the signal ampplification process. We sometimes : use qPCR for about 100 cells with good sensitivity. : : cycle
What you said is true for absolute quantification. However, most people are only interested in relative quantification focusing on expression changes ( in percentage term). In this case, a reference RNA sample is included for comparison, and the "anomaly" from the first few cycles should have been cancelled out. In our experience, replicated reactions should always have <0 .2 cycle difference. And a >0.4 (30% change) cycle change can be reliably detected.
But any detection system would have the same issue. For example, you can never say that all RNA molecules are hybridized with the same sensitivity and specificity using nannostring probes. Actually, qPCR is considered more unbiased compared to probe hybridization.
【在 l***s 的大作中提到】 : But any detection system would have the same issue. For example, you can : never say that all RNA molecules are hybridized with the same sensitivity : and specificity using nannostring probes. Actually, qPCR is considered more : unbiased compared to probe hybridization.
ok. 有人做过就行。 记得有篇用这个screen ChIP-seq antibody的paper, 我觉得就是很好的应用。 有一些locus一直要反复看,虽然第一次成本很高,但是滩平了很便宜快速
【在 r*m 的大作中提到】 : 我做过300个基因的probeset,没有问题,很好很强大。
l*e
81 楼
This is helpful, Thanks a lot!!
nanostring
【在 l***s 的大作中提到】 : I did some comparative analysis using our own qPCR, RNA-seq, and nanostring : data. Looks like qPCR is still the best. Of course, it is a challenge to : analyze several hundred genes using qPCR. Typically, we still need to : validate nanostring data using an independent approach such as qPCR.