Ribosome-RNA-removed reverse transcription primer pool, kit and method for removing ribosome RNA
A technology of reverse transcription primers and ribosomes, applied in DNA/RNA fragments, recombinant DNA technology, chemical libraries, etc., can solve the problems of data redundancy, high price, affecting the detection and analysis of mRNA or LncRNA, etc. The effect of application range
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Embodiment 1
[0061] 1. Reverse transcription primer design and rRNA removal experiment
[0062] 1. Plant cultivation
[0063] The cooperative laboratory cultivates and collects Arabidopsis plant tissues through tissue culture.
[0064] 2. Total RNA extraction
[0065] Follow the instructions of Qiagen's QIAamp RNA RNeasy Mini Kit for total RNA extraction, starting with 1 μg of total RNA for subsequent reactions.
[0066] 3. Mix reverse transcription primers into a reverse transcription primer pool
[0067] Design and synthesis of reverse transcription primers. The reverse transcription is designed on the RNA sequence of rRNA (NC_003071.7) according to the design density of 500bp / strand, 1000bp / strand and 2000bp / strand. The mixed reverse transcription primer is RT Primer pool, Each reverse transcription primer was diluted to 10μM with sterile water.
[0068] Design density of 1000bp / bar (denoted as RT-1000bp group), corresponding to the pool of 5 reverse transcription primers that are ticked in 1Kbp / i...
Embodiment 2
[0107] Example 2 Exploring the concentration of reverse transcription primers
[0108] 1. Plant cultivation: the specific method is the same as in Example 1.
[0109] 2. Total RNA extraction: The specific method is the same as in Example 1, and the initial amount of total RNA is 1 μg.
[0110] 3. Mixing of reverse transcription primers
[0111] The reverse transcription primers are designed for reverse transcription with a design density of 1000bp / strand. The mixed reverse transcription primers are RT Primerpool, and each reverse transcription primer is diluted with sterile water to 5μM, 10μM, 50μM and 100μM. Corresponding to the pool of 5 reverse transcription primers that are ticked in the 1000bp / item in Table 1, each reverse transcription primer is complementary to a region of the precursor RNA sequence. The concentration of any two reverse transcription primers in the reverse transcription primer pool is equal. The reverse transcription primers were synthesized by Shanghai Jieru...
Embodiment 3
[0123] Example 3 Exploration of RNase H and DNase I dosage
[0124] 1. Plant cultivation: the specific method is the same as in Example 1.
[0125] 2. Total RNA extraction: The specific method is the same as in Example 1, and the initial amount of total RNA is 1 μg.
[0126] 3. Mixing of reverse transcription primers
[0127] The reverse transcription primers are designed for reverse transcription with a design density of 1000 bp / strand. The mixed reverse transcription primers are RT Primerpool, and each reverse transcription primer is diluted to 10 μM with sterile water. Corresponding to the pool of 5 reverse transcription primers that are ticked in the 1000bp / item in Table 1, each reverse transcription primer is complementary to a region of the precursor RNA sequence. The concentration of any two reverse transcription primers in the reverse transcription primer pool is equal. The reverse transcription primers were synthesized by Shanghai Jierui Biological Engineering Co., Ltd.
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