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求miRNA表达水平检测具体方法
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| 有一种荧光定量PCR检测的方法，是利用茎环引物做的，想知道具体方法，论文形式最好，越具体越好 |
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bomuwang
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2楼2009-12-25 19:32:37
3楼2009-12-25 21:32:43
telomerase
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4楼2009-12-26 03:49:57
telomerase
至尊木虫 (著名写手)
cain
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★ ★ ★
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Real-time RT-PCR for miRNA detection 187-plex RT-PCR was adapted for the rat sequences from Lao et al. (2006) based on Sanger’s October 2005 registry. Briefly, multiplex reverse transcription primers were generated with a common 20-nt stem–loop followed by 8 nt complementary to the 39-end of a given miRNA (e.g., 59-AACTATAC-39 for rnolet- 7a). Reverse transcriptase reactions were performed in 5 mL containing 13 cDNA Archiving Kit buffer (Applied Biosystems), 10 units of MMLV reverse transcriptase, 1.25 mM each dNTP, 1.3 units of RNase inhibitors (Applied Biosystems), 2.5 nM each reverse primer, and 2 mL of laser capture microdissection samples. Reactions were 30 cycles for 30 sec at 20°C, 30 sec at 42°C, and 1 sec at 50°C and one cycle for 5 min at 85°C to inactivate the enzyme. Pre-PCR cDNA was pre-amplified using a common reverse primer (UR) to the 20-nt stem–loop and multiplexed z18-nt forward primers (FPs) with a common 59-end and a 39-end matching 12–17 nt of the 59-end of a given miRNA (e.g., 59-TCCAGCTCCTATATGAT- 39 for rno-let-7a). The amplification was performed in 25 mL containing 13 Universal Master Mix with no UNG (Applied Biosystems), 50 nM each FP, 5 mM UR, 6.25 units of AmpliTaq Gold polymerase (Applied Biosystems), 0.5 mM each dNTP, 2 mM MgCl2, and 5 mL of the initial reverse transcriptase amplification. Reactions were one cycle of 10 min at 95°C, 2 min at 55°C, and 14 cycles of 1 sec at 95°C and 1 min at 65°C. Real-time PCR The product of this amplification was diluted four times, and 0.1 mL was used for real-time PCR performed in 10 mL containing 13 Universal Master Mix with no UNG (Applied Biosystems), 0.25 mM FP, 0.1 mM TaqMan probe, and 5 mM UR. The miRNA specificity for each reaction was provided by the miRNA-specific sequences of the FP and TaqMan probes. Real-time PCR was performed in a Biosystems 7500HT 96-well plate Sequence Detection System using 40 cycles of 15 sec at 95°C and 1 min at 60°C. All reactions were run in duplicate. The threshold cycle (Ct) was determined as the fractional cycle number at which the fluorescence passes the fixed threshold. For primer sequences, see Supplemental Table 2. All Ct values were averaged over two to four repeated biological experiments each with two averaged realtime PCR experimental replicates. Single-plex real-time PCR Single-plex real-time PCR was performed as described for multiplex real-time PCR with one modification: A single miRNAspecific primer was used instead of multiple primers. Real-time PCR for mRNA detection RT-PCR was performed to measure the amount of mRNAs in the same laser capture microdissected samples. Reverse transcriptase reactions were performed in 5 mL containing 13 cDNA Archiving Kit buffer (Applied Biosystems), 10 units MMLV reverse transcriptase, 1.25 mM each dNTP, 1 unit of RNase inhibitor (Applied Biosystems), 2 mM random primer, and 1 mL of laser capture microdissection samples. Reactions were 10 min at 20°C, 120 min at 37°C, and 5 min at 85°C to inactivate the enzyme. cDNA was pre-amplified using gene-specific forward primers and reverse primers. The amplification was performed in 25 mL containing 13 Universal Master Mix with no UNG (Applied Biosystems), 50 nM each FP, 50 nM each RP, 6.25 units of AmpliTaq Gold polymerase (Applied Biosystems), 0.5 mM each dNTP, 0.75 mM MgCl2, and 5 mL of the initial reverse transcriptase amplification. Reactions were one cycle for 10 min at 95°C, 2 min at 55°C, and 14 cycles of 1 sec at 95°C and 1 min at 65°C. The product of this amplification was diluted four times, and 0.1 mL was used for realtime PCR reactions detecting mRNAs. It was performed in 10 mL coBiosystems), 500 nM FP, 200 nM TaqMan probe, and 500 nM UR. Real-time PCR was performed as above. For primer sequences, see Supplemental Table 1.ntaining 13 Universal Master Mix with no UNG (Applied |
5楼2009-12-26 03:52:20