PMID- 31081819
OWN - NLM
STAT- MEDLINE
DCOM- 20200318
LR  - 20200318
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 146
DP  - 2019 Apr 24
TI  - Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify 
      and Localize mRNAs in Murine Oocytes.
LID - 10.3791/59414 [doi]
AB  - Current methods routinely used to quantify mRNA in oocytes and embryos include 
      digital reverse-transcription polymerase chain reaction (dPCR), quantitative, 
      real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are 
      performed using a single oocyte or embryo, low-copy mRNAs are not reliably 
      detected. To overcome this problem, oocytes or embryos can be pooled together for 
      analysis; however, this often leads to high variability amongst samples. In this 
      protocol, we describe the use of fluorescence in situ hybridization (FISH) using 
      branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in 
      individual cells. When the technique is coupled with Spot Finding and 
      Tracking computer software, the abundance of mRNAs in the cell can also be 
      quantified. Using this technique, there is reduced variability within an 
      experimental group and fewer oocytes and embryos are required to detect 
      significant differences between experimental groups. Commercially available 
      branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned 
      tissues or adherent cells on slides. However, oocytes do not effectively adhere 
      to slides and some reagents in the kit were too harsh resulting in oocyte lysis. 
      To prevent this lysis, several modifications were made to the FISH kit. 
      Specifically, oocyte permeabilization and wash buffers designed for the 
      immunofluorescence of oocytes and embryos replaced the proprietary buffers. The 
      permeabilization, washes, and incubations with probes and amplifier were 
      performed in 6-well plates and oocytes were placed on slides at the end of the 
      protocol using mounting media. These modifications were able to overcome the 
      limitations of the commercially available kit, in particular, the oocyte lysis. 
      To accurately and reproducibly count the number of mRNAs in individual oocytes, 
      computer software was used. Together, this protocol represents an alternative to 
      PCR and sequencing to compare the expression of specific transcripts in single 
      cells.
FAU - Timme, Kelsey R
AU  - Timme KR
AD  - Department of Animal Science, University of Nebraska-Lincoln.
FAU - Wood, Jennifer R
AU  - Wood JR
AD  - Department of Animal Science, University of Nebraska-Lincoln; jwood5@unl.edu.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20190424
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (RNA, Messenger)
SB  - IM
MH  - Animals
MH  - Embryo, Mammalian/cytology
MH  - Female
MH  - *In Situ Hybridization, Fluorescence
MH  - Mice
MH  - Oocytes/cytology/*metabolism
MH  - Polymerase Chain Reaction
MH  - RNA, Messenger/metabolism
EDAT- 2019/05/14 06:00
MHDA- 2020/03/19 06:00
CRDT- 2019/05/14 06:00
PHST- 2019/05/14 06:00 [entrez]
PHST- 2019/05/14 06:00 [pubmed]
PHST- 2020/03/19 06:00 [medline]
AID - 10.3791/59414 [doi]
PST - epublish
SO  - J Vis Exp. 2019 Apr 24;(146). doi: 10.3791/59414.