Measurement of telomere length in samples fixed in formalin, embedded in paraffin (FFPE) by quantitative PCR

摘要

A method for measuring telomere length in a formalin-fixed paraffin embedded tissue comprising: (a) extracting the target telomeric nucleic acid from a paraffin-embedded tissue, formalin fixed using a gentle extraction method that does not isolate the fragments DNA in a column and that retains a majority of telomeric target nucleic acid fragments that are at least 50 bp, (b) combining in a polymerase chain reaction the telomeric target nucleic acid extracted from step (a) comprising substantially a first and a second complementary chain, a first telomeric primer in which the first telomeric primer is capable of hybridizing with the first chain of the target telomeric nucleic acid and being extended by DNA polymerase to form an extended telomeric primer, and a second telomeric primer, in which the second telomeric primer is capable of hybridizing with the first extended telomeric primer C and to be extended by DNA polymerase but is not capable of hybridizing with the target telomeric nucleic acid and which is extended by DNA polymerase; (c) heating the polymerase chain reaction to a temperature sufficient to generate nucleic acid mono chains and reduce the temperature to a temperature between 49 ° C and 60 ° C to allow the primers to hybridize with their targets and spread , and (d) repeat step (c) for at least 4 cycles, (e) heat the polymerase chain reaction to a temperature sufficient to generate a single chain of nucleic acids and reduce the temperature from 55 ° C to 65 ° C to allow primers to hybridize with their targets and extend for another 20 to 40 more cycles, (f) identify the replication cycle in which the threshold PCR signal is passed, and (g) determine the length of medium telomere.