Ribonucleic acid quantification for the performance of RT- PCR technique

Revista Cubana de Hematología, Inmunología y Hemoterapia

Suárez-González, Yandi¹  González-Mugica Romero, Raúl¹  Amor-Vigil, Ana María¹  Garrote-Santana, Heidys¹  Díaz-Alonso, Carmen¹ 

[1] Instituto de Hematología e Inmunología, La Habana, Cuba

关键词: molecular biology;    PCR;    RNA quantification;    RT-PCR;   

DOI  :  

学科分类：生理学与病理学

来源: SciELO

【 摘 要 】

The polymerase chain reaction (PCR) is a molecular biology technique which aim is to obtain a great number of copies of a fragment of deoxyribonucleic acid (DNA). Reverse transcription polymerase chain reaction (RT-PCR) is one variant of the conventional PCR, in which ribonucleic acid (RNA) is used as a pattern to synthesize the complementary DNA (DNAc), required for the following PCR technique. The RNA is a material susceptible to degradation due to the action of ribonucleases. In general, a very little quantity is obtained, hence, its quantification would lead to a loss of an appreciable part of it. Conventional spectrophotometers use large volume samples and although it is possible to perform a dilution, it sometimes can result in the risk of not detecting the concentration. Medullary blood from ten patients with different hematologic diseases was studied. Aspirate sample volume varied between 5 to 8 mL. The extraction and purification of RNA was carried out manually. The quantification was performed in a Multi-volume Spectrophotometer using 2 µL sample volumes. Optical density reading was performed at 260 and 280 nm to measure nucleic acids and proteins, respectively. RNA integrity was analyzed by means of agarose horizontal gel electrophoresis on 2 % with ethidium bromide as fluorescent marker. Concentrations varied from 444,4 to 2515,5 ng/µL. Purity rate varied between 1,111 and 2,091. No total degradation of the samples was observed. There are numerous factors which can affect the RNA performance and preservation, thus, the integral analysis of its amount and quality are essential to successfully carry out the reverse transcription and the following PCRs.

【 授权许可】

CC BY-NC