Record Details

Total RNA content in sheep oocytes and developing embryos produced in vitro, a comparative study between spectrophotometric and fluorometric assay

KRISHI: Publication and Data Inventory Repository

View Archive Info
 
 
Field Value
 
Title Total RNA content in sheep oocytes and developing embryos produced in vitro, a comparative study between spectrophotometric and fluorometric assay
Not Available
 
Creator Mishra A
Reddy IJ
Gupta PSP
Mondal S
 
Subject RNA
oocytes
embryos
sheep
spectrophotometer
fluorometer
 
Description Not Available
Isolation of total RNA from limited number of oocytes and embryos is a big challenge. DNA free RNA and assessment of RNA integrity are crucial to the success of gene expression studies because poor quality RNA give misleading results. The objective of the present study was to establish a suitable protocol to isolate good quality total RNA from a minimal number of sheep oocytes and embryos that enables the downstream applications, as well as to estimate RNA content in oocytes and developmental stages of embryos. Five protocols were approached to isolate total RNA from oocytes and embryos. Four methods were by standard Trizol protocols and its modification whereas fifth method was by commercial kit (RNeasy mini kit, Quiagen). Total RNA isolated by modified Trizol protocol with coprecipitants (acrylamide and glycogen) showed significantly (P < 0.05) more spectrophotometric reading of RNA concentration than by modified Trizol protocol without coprecipitant followed by commercial kit and conventional Trizol protocol. RNA quality, purity, concentration, RNA per oocyte and expression of GAPDH (house keeping gene) were compared to find the best RNA isolated by different protocols. Spectrophotometric and fluorometric assay were compared to quantify the total RNA concentration in sheep oocytes and different stages of developing embryos. RNA yield by spectrophotometer analysis showed 5–100 times more reading than fluorometer. Significant (P < 0.05) reduction in RNA content was observed in matured oocytes than that of immature oocytes. There was significant (P < 0.05) increase in RNA content after fertilization upto 2–4 cells stage followed by significant (P < 0.05) decrease at 8–16 cells and increased at morula. RNA concentration at blastocyst was significantly low than at morula. From the protocols approached modified Trizol protocol with coprecipitant was most efficient and suitable method over other protocols approached to isolate RNA from few sheep oocytes and embryos for gene expression study.
Not Available
 
Date 2018-11-22T10:22:25Z
2018-11-22T10:22:25Z
2018-02-02
 
Type Research Paper
 
Identifier Mishra A, Reddy IJ, Gupta PSP and Mondal S. 2018. Total RNA content in sheep oocytes and developing embryos produced in vitro, a comparative study between spectrophotometric and fluorometric assay. Cytology and Genetics, 52:62-74.
Not Available
http://krishi.icar.gov.in/jspui/handle/123456789/12811
 
Language English
 
Relation Not Available;
 
Publisher Springer Link