RNA interference, RNA isolation and introduction to RT-PCR Flashcards
Total RNA is a term to describe what?
The entire complement of RNA molecules found in any given cell and includes the three major types of RNA molecules:
1- mRNA
2- tRNA
3- rRNA
______ is transcribed as a ______-stranded molecule containing ________ from gene-containing template of DNA.
1 - mRNA
2 - single
3 - ribonucleotdies
____ is involved in protein ________ within the ribosome (composed of _____), and functions by converting the information carried by the ______ into a corresponding _____ _____ sequence.
1 - tRNA 2 - synthesis 3 - rRNA 4 - mRNA 5 - amino acid
What are examples of small RNA molecules that play a variety of roles in regulatory function?
snRNA - small nuclear RNA
miRNA - micro RNA
What is snRNA involved in?
Involved in the maturation of mRNA.
What is miRNA involved in?
The regulation of gene expression.
mRNA is the key link between the information stored within a _____ and the expression of that information via ________ _________.
1 - gene
2 - protein synthesis
What is the central dogma of molecular biology?
DNA –> mRNA –> protein
mRNAs serve a central function in what?
Transport, regulation and translation of information from DNA nucleotides to a sequence of aa’s that ultimately form fully functional proteins.
mRNA expression is controlled by a number of regulatory proteins to regulate many events that affect the cell cycle such as?
1 - level and/or stability of mRNA in specific cell types
2 - ability to form functional proteins following post-transcriptional modification
3 - mRNA translocation in the cell
In this lab, we will be investigating which technique?
RNAi
The ____ process of RNAi was originally discovered in ______, where it was originally described as what?
1 - in-vivo
2 - plants
3 - post-transcriptional gene silencing
RNAi is a powerful tool used to what?
Artificially down-regulate the expression of specific target genes.
RNAi is a natural process. How so?
Natural process triggered by dsRNA precursors that are present during viral infection of mammalian systems.
Precursors are processed into what, with a length of what? (dsRNA precursors from viral infection)
Short RNA duplexes of 21-28 nucleotides in length.
siRNAs consist of _____ bp duplexes, containing both the ____ and ____-_____ strands with ___ ______ that overhang at the __ end.
1 - 19-21
2 - sense and anti-sense
3 - two nucleotides
4 - 3’
siRNAs are translocated into mammalian cells using a _______ _______ containing a combination of _________ that allow for the transfection of small RNA molecules into the cytoplasm without _______ impacts on the cells.
1 - transfection reagent
2 - polyamines
3 - cytotoxic
What do siRNA molecules do upon entering the cell?
Bind to a nuclease complex to form the RISC complex.
The RISC complex is ___-dependent.
ATP
Following activation of the RISC complex, the siRNA molecule _______.
unwinds
The conjoined siRNA molecule and nuclease complex targets the _________ transcript (i.e. __________ ___ strand) by base pair matching.
1 - homologous
2 - complementary RNA
This affinity to complementary mRNA allows for the ____-specific ________ of targeted _____ molecules by the RISC complex.
1 - gene
2 - splicing
3 - mRNA
The mRNA is cleaved approximately _____ base pairs from the ___ end of the siRNA molecule, thereby causing a reduction in _____.
1 - 12-15
2 - 3’
3 - gene expression levels
What is the first task in this lab?
Isolate total RNA from hek293 cells that have been treated with either 25 nM siTALIN, 25nM siGADPH or 25nM negative control siRNA for 48 hours.
What are the factors that must be considered in order to obtain the expected results?
1 - RNA contains an additional OH in the ribose sugar making it more susceptible to hydrolysis and thus degradation
2 - RNA is ss and thus less protected than DNA
3 - RNase enzymes are abundant in the lab environment
What has been done to reduce the possibility of RNase contamination?
All solutions, glassware, and plastic-ware will be pre-treated to denature/destroy ribonucleases.
What are the solutions pre-treated with?
DEPC which inactivates ribonucleases.
What buffers cannot be treated wit DEPC?
Tris based solutions.
The ______ is unable to destroy RNases so, glassware will be?
1 - autoclave
2 - baked at 400C for at least 4 hrs
Tips and tubes are certified _____ free.
RNase
Why are gloves so important?
Major source of RNase.
What will we be using to isolate RNA from 293 cells?
RNease Mini kit that is produced by QIAGEN.
Following RNA isolation, we will generate ____, and perform _____ to determine the expression levels of ______ and ______ from the siRNA treated cells.
1 - cDNA
2 - pcr
3 - GAPDH and TALIN
