Reverse transcription primer pool for removing rRNA, kit and method for removing rRNA
A technology of reverse transcription primers and ribosomes, applied in biochemical equipment and methods, DNA/RNA fragments, DNA preparation, etc., can solve data redundancy, affect the detection and analysis of mRNA or LncRNA, rRNA residues, etc. To achieve the effect of broadening the scope of application
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Embodiment 1
[0061] 1. Reverse transcription primer design and rRNA removal experiment
[0062] 1. Cell culture
[0063] Use GIBCO's DMEM and 10% FBS serum at 37°C, 5% CO 2 The 293T cells were cultured under culture conditions, and after 2 days of normal culture, the cell density was about 60-80% (40,000-50,000).
[0064] 2. Total RNA extraction
[0065] Total RNA was extracted according to Qiagen's QIAamp RNA RNeasy Mini Kit instructions, starting with 1 μg of total RNA for subsequent reactions.
[0066] 3. The reverse transcription primers are mixed into a reverse transcription primer pool
[0067] Design and synthesis of reverse transcription primers, design reverse transcription primers on the precursor RNA 45S sequence of rRNA (sequence number: NR_046235.1) according to the design density of 500bp / , 1000bp / , 2000bp / and 5000bp / product , the mixed reverse transcription primer is RT Primer pool (abbreviated as reverse transcription primer pool), and each reverse transcription primer...
Embodiment 2
[0107] The concentration exploration of embodiment 2 reverse transcription primers
[0108] 1. Cell culture: the specific method is the same as in Example 1.
[0109] 2. Extraction of total RNA: the specific method is the same as in Example 1, and the initial amount of total RNA is 1 μg.
[0110] 3. Reverse transcription primer mix
[0111] The reverse transcription primers were designed for reverse transcription at a design density of 1000bp / strip. The mixed reverse transcription primers were RT Primerpool, and each reverse transcription primer was diluted to 5 μM, 10 μM, 50 μM and 100 μM with sterile water. A pool formed by mixing a total of 12 reverse transcription primers corresponding to all ticked 1000bp / entry items in Table 1, each reverse transcription primer is complementary to a region on the precursor RNA 45S sequence. The concentrations of any two reverse transcription primers in the reverse transcription primer pool are equal. The reverse transcription primers ...
Embodiment 3
[0124] Embodiment 3 RNase H and DNase I dosage exploration
[0125] 1. Cell culture: the specific method is the same as in Example 1.
[0126] 2. Extraction of total RNA: the specific method is the same as in Example 1, and the initial amount of total RNA is 1 μg.
[0127] 3. Reverse transcription primer mix
[0128] The reverse transcription primers were designed for reverse transcription at a design density of 1000bp / strip. The mixed reverse transcription primers were RT Primerpool, and each reverse transcription primer was diluted to 10 μM with sterile water. A pool formed by mixing a total of 12 reverse transcription primers corresponding to all ticked items of 1000 bp / item in Table 1, each reverse transcription primer is complementary to a region on the precursor RNA 45S sequence. The concentrations of any two reverse transcription primers in the reverse transcription primer pool are equal. The reverse transcription primers were synthesized by Shanghai Jierui Bioengine...
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