Determination method of cytokeratin 19 mRNA
A technique for measuring cytokeratin and its method, which is applied in the field of reagents for measuring cytokeratin 19 mRNA, and can solve problems such as difficulty in primers
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Embodiment 1
[0115] The preparation of embodiment 1 standard RNA
[0116] CK19 RNA (hereinafter referred to as standard RNA) used in Examples described later was prepared by the methods shown in (1) to (2).
[0117] (1) Double-stranded DNA from bases 160 to 1353 (1194 bases) in the base sequence of CK19 registered in GenBank (GenBank Accession No. NM_002276, 1490 bases) was cloned.
[0118] (2) Using the double-stranded DNA prepared in (1) as a template, perform in vitro transcription. The double-stranded DNA was then completely digested by DNase I treatment, followed by purification and preparation of RNA. The RNA was quantified by measuring the absorbance at 260 nm.
Embodiment 2
[0119] Example 2 Preparation of intercalated fluorochrome-labeled oligonucleotide probes
[0120] An oligonucleotide probe labeled with an intercalating fluorochrome is prepared. Amino groups were introduced into T at the ninth position from the 5' end of the sequence described in SEQ ID NO: 39 and T at the 12th position from the 5' end of the sequence described in SEQ ID NO: 40 using Label-ON Reagents (manufactured by Clontech Corporation). , A at the 13th position from the 5' end of the sequence described in SEQ ID NO: 41, C at the 13th position from the 5' end of the sequence described in SEQ ID NO: 42, and the 5' from the sequence described in SEQ ID NO: 43 T at the 7th position from the end, G at the 11th position from the 5' end of the sequence described in SEQ ID NO: 44, C at the 10th position from the 5' end of the sequence described in SEQ ID NO: 45, and SEQ ID NO: 46 The G position at the ninth position from the 5' end of the described sequence was further modified ...
Embodiment 3
[0121] Example 3 Determination of CK19 RNA (Part 1)
[0122]Using the cleavage oligonucleotide shown in [1] to [28] of the combinations shown in Table 1, the first primer, the second primer, and the intercalating fluorescent dye-labeled oligonucleotide probe (hereinafter referred to as INAF Probe), by the method shown in (1) to (4), carry out the determination of standard RNA. It should be noted that in Table 1, serial numbers 19 to 22 are partial sequences of serial number 1, serial numbers 23 and 24 are partial sequences of serial number 2, serial numbers 25 and 26 are partial sequences of serial number 3, and serial numbers 27 and 28 are partial sequences of serial number 4, serial numbers 29 to 32 are partial sequences of serial number 5, serial numbers 33 and 34 are partial sequences of serial number 6, serial numbers 35 and 36 are partial sequences of serial number 7, Serial numbers 37 and 38 are partial sequences of serial number 8.
[0123] Table 1
[0124]
[01...
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