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【求助/交流】real-time PCR cDNA模板问题
看了不少资料，还是不明白做实时定量PCR时，起始的cDNA模板必须是一致的吗?我目前用的是从1ugRNA中得到的，直接用于实时定量，但用完了。我第一次做反转录时，不知道必须用等量的RNA，所以只是取了1ulRNA 反转录。现在用的cDNA用完了，不知道以前这种方式得到的cDNA是否将浓度调到一致后可以使用？多谢答复。
3，反转成cDNA，这时候要根据你上一步测的浓度，在反转录中加入同样质量的RNA,一般为一个反应反转录250ng,或者500ng的RNA，
4，反转录完成后，稀释5倍或10倍，取2UL做模板做QRT-PCR，最好是CT值落在线性范围内，CT值不要太早或者太晚出来，二十多比较好。
我觉得我说的应该比较清楚了吧。:tiger24: Originally posted by 彬心玉壶 at
3，反转成cDNA，这时候要根据你上一步测的浓度，在反转录中加入同样质量的RNA,一般为一个反应反转录250ng,或者500ng的RNA，
4，反转录完成后，稀释5倍或10倍，取2UL做模板做QRT-PCR，最好是 ... [/
我就是不明白加内标不就是为了均一化模板吗？为什么还需要使用等量的RNA 呢？ 不必，有你的内参呢 将RNA浓度定量到1ug进行反转录，这样得到的cDNA可以认为是均一的，其进行荧光定量PCR时扩增曲线好看，并且较易分析，内参当然是必须的了，所以很能说明问题。
这也只是我所了解到的，希望与大家共同交流。 Originally posted by gdongli8325 at
Originally posted by 彬心玉壶 at
我就是不明白加内标不就是为了均一化模板吗？为什么还需要使用等量的RNA 呢？ 你说的内标是指的什么？
我做过绝对定量和相对定量，在相对定量作基因表达量变化的时候，是要用管家基因作为参照基因。
在定量的过程中，每个样品（没处理和处理过的样品，就像经常举的例子一样，正常细胞和癌变细胞）都会用管家基因做对照的。
你的问题中有点我都没看明白：我第一次做反转录时，不知道必须用等量的RNA，所以只是取了1ulRNA 反转录。
你取了1ul反转录，貌似没法定量吧 因为不知道浓度。 我们做相对定量，看基因的表达量情况是建立在假定RNA它的反转录效率是一样的。
我没有做过先反转录成CDNA，在定量CDNA的量去做模板。可能方法不一样吧，具体的我就不知道了。 Originally posted by gaozx123 at
将RNA浓度定量到1ug进行反转录，这样得到的cDNA可以认为是均一的，其进行荧光定量PCR时扩增曲线好看，并且较易分析，内参当然是必须的了，所以很能说明问题。
这也只是我所了解到的，希望与大家共同交流。 我们说的应该是一样的，只是我先将RNA定量到250或者500ug进行反转录进行反转录，然后再稀释而已。
其实都是建立在：反转录效率是一样的情况下，（其实不一样，所有现在很多都用好几个管家基因进行定量，减小误差） 同意楼上，用反转录成的cDNA去定量其实是不准确的，可能我们用的是总RNA，所以只能定量RNA浓度之后再反转。
楼主所说的定量cDNA，我想应该是半定量吧，那样调出来的浓度是根据条带的灰度值调的，并不是准确定量。最好还是用RNA定量后反转。 LZ的问题没太看明白，不过我也一直在做这个。real time-PCR本身就是一个定量的反应，所以在cDNA转录的时候一定要定量，一般都是加1ug的RNA。然后在PCR的时候把模板适当稀释。当然内参也是必须的~
var cpro_id = 'u1216994';
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PP4R2,GHRHR and clusterin are significantly highly expressed in prolactinoma,somatotroph and nonfunctioning adenomas respectively,which was also proved by RT-PCR and realtime-PCR.
PP4R2,GHRHR和clusterin分别在泌乳素瘤,生长激素瘤,无激素瘤中明显高表达,并经RTPCR,实时定量PCR等证实。
Methods The differentially expressed genes in pituitary adenomas were studied by complementary DNA microarrays, and confirmed by RT-PCR and realtime-PCR.
方法 用cDNA微阵列技术筛选垂体瘤组织差异表达基因,对于在三种垂体瘤中差异表达基因用RT-PCR和实时定量PCR进行初步验证。
Real Time- PCR and Western blotting were used to detect the different expression of PSD-95 mRNA and protein levels after SCI.
采用实时定量PCR和Western印迹法测定损伤后各时间段PSD-95 mRNA及其蛋白水平在脊髓中的表达变化。
Methods Retrovirus infection method was employed to develop an immortalized rat astrocyte strain expression enkephalin regulated by doxycycline. hPPE gene expression level of IAST/Tet-On/hPPE strain was detected by Real time-PCR and radioimmunoassay.
方法应用逆转录病毒转染法,建立受强力霉素调控表达人前脑啡肽原基因(hPPE)的永生化大鼠星形胶质细胞株(IAST/Tet-On/hPPE),实时定量PCR及放射免疫分析法检测强力霉素对该细胞株脑啡肽表达的定量调节。
Methods The differentially expressed genes in pituitary adenomas were studied by complementary DNA microarrays,and confirmed by RT-PCR and realtime-PCR.
方法用cDNA微阵列技术筛选垂体瘤组织差异表达基因,对于在3种垂体瘤中高差异表达基因用RTPCR和实时定量PCR进行初步验证。
PP4R2,GHRHR and clusterin are significantly highly expressed in prolactinoma,somatotroph and nonfunctioning adenomas respectively,which was also proved by RT-PCR and realtime-PCR.
PP4R2,GHRHR和clusterin分别在泌乳素瘤,生长激素瘤,无激素瘤中明显高表达,并经RTPCR,实时定量PCR等证实。
Methods The differentially expressed genes in pituitary adenomas were studied by complementary DNA microarrays, and confirmed by RT-PCR and realtime-PCR.
方法 用cDNA微阵列技术筛选垂体瘤组织差异表达基因,对于在三种垂体瘤中差异表达基因用RT-PCR和实时定量PCR进行初步验证。
Real Time- PCR and Western blotting were used to detect the different expression of PSD-95 mRNA and protein levels after SCI.
采用实时定量PCR和Western印迹法测定损伤后各时间段PSD-95 mRNA及其蛋白水平在脊髓中的表达变化。
Methods Retrovirus infection method was employed to develop an immortalized rat astrocyte strain expression enkephalin regulated by doxycycline. hPPE gene expression level of IAST/Tet-On/hPPE strain was detected by Real time-PCR and radioimmunoassay.
方法应用逆转录病毒转染法,建立受强力霉素调控表达人前脑啡肽原基因(hPPE)的永生化大鼠星形胶质细胞株(IAST/Tet-On/hPPE),实时定量PCR及放射免疫分析法检测强力霉素对该细胞株脑啡肽表达的定量调节。
Methods The differentially expressed genes in pituitary adenomas were studied by complementary DNA microarrays,and confirmed by RT-PCR and realtime-PCR.
方法用cDNA微阵列技术筛选垂体瘤组织差异表达基因,对于在3种垂体瘤中高差异表达基因用RTPCR和实时定量PCR进行初步验证。
The expression of IFN-γ mRNA,IL-10 mRNA and IL-12 mRNA in PBMC were detected by real time-PCR.
用实时足量聚合酶链反应(real time-PCR)法检测PBMC IFN-γmRNAI、L-12 mRNA和IL-10 mRNA的表达。
Real time-PCR was used to determine ob, Ob-Rl, IGF-1 and PPARγ genes expression.
合成D6、Ob-Rl、IGF-1和PPARγ引物,利用Real-time PCR检测不同部位脂肪组织中这4种基因的mRNA水平。
Sixteen hours post-infection, HBV contained in AD38 cells were released and AD38 culture medium was harvested every two hours and the HBV contained in AD38 cells were determined by realtime-PCR.
所获得的重组病毒以不同M．O．I感染含HBV的AD38细胞，过夜培养约16小时后，释放AD38内的HBV，收获AD38细胞培养液，qRealtime-PCR法检测其中HBV(HBV基因组内含WPRE元件)含量，同时用重组AAV／EGFP病毒做对照。
The TFR mRNA expression was determined by realtime-PCR method.
方法以紫外线(UVB)照射HaCat,流式细胞仪检测TFR的表达,realtime-PCR检测TFRmRNA表达。
the PPARγmRNA in liver by real time-PCR, cytokine(IL-2 、 IL-10) levels in plasma were also measured with enzyme linke
(3)用荧光定量PCR方法行肝PPARγmRNA测定； ELISA法测定血浆中细胞因子IL-2、IL-10的变化；
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&2008中国知网(cnki) 中国学术期刊(光盘版)电子杂志社Quantitative PCR (qPCR) |
Real-Time PCR, also referred to as Quantitative PCR (or qPCR), was developed as a precise, efficient and rapid method for nucleic acid detection. This technique is based on traditional Polymerase Chain Reaction (PCR) technology with a few improvements:
Real-Time PCR combines nucleic acid amplification and detection in one step
It can use smaller amounts of starting material than traditional PCR
It is possible to quantify the product based on fluorescent detection
There is no post-amplification process
The detection of template DNA (or RNA if a reverse transcription step is added prior to amplification) is live and based on when the PCR product is amplified above the threshold of background or the cycle threshold number (Ct number). Far different from the former end-point method of standard PCR techniques, Real-Time PCR can be quantitative because the PCR product is detected using fluorescent dyes in real time.
Thus, Real-Time PCR earned the alternate and distinctive name of Quantitative PCR or qPCR.
Key Applications of Real-Time PCR:
qPCR has been used for a wide number of applications over the years but there some uses for the technique that have been particularly popular.
These include:
Gene Expression (mRNA) Analysis
microRNA and Non-Coding RNA analysis
Genetic Variation
Mutation Detection
SNP Analysis
Genotyping/Allelic Discrimination
Real-Time Instruments:
When it comes to instrumentation, purchasing a Real-Time Thermocycler can be a significant investment.
Things to consider before purchasing a qPCR machine include the overall footprint, the block formats available, the run times, expandability, the software used, volume limits for wells, etc.
You can compare specifications on some of the latest instruments available here:
Range of Real-Time PCR Kits and Reagents:
As with most techniques there are now a number of ready-made kits available to make qPCR easier and more reproducible.
can be purchased which include all reagents needed for a successful reaction.
These kits usually include everything you need to run a qPCR reaction except the primers for your particular gene of interest.
Individual components c this includes everything from ,
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