RNA interference, RNA isolation and introduction to RT-PCR

Question 1

Total RNA is a term to describe what?

Answer 1

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

Question 2

______ is transcribed as a ______-stranded molecule containing ________ from gene-containing template of DNA.

Answer 2

1 - mRNA
2 - single
3 - ribonucleotdies

Question 3

____ is involved in protein ________ within the ribosome (composed of _____), and functions by converting the information carried by the ______ into a corresponding _____ _____ sequence.

Answer 3

1 - tRNA
2 - synthesis
3 - rRNA
4 - mRNA
5 -  amino acid

Question 4

What are examples of small RNA molecules that play a variety of roles in regulatory function?

Answer 4

snRNA - small nuclear RNA

miRNA - micro RNA

Question 5

What is snRNA involved in?

Answer 5

Involved in the maturation of mRNA.

Question 6

What is miRNA involved in?

Answer 6

The regulation of gene expression.

Question 7

mRNA is the key link between the information stored within a _____ and the expression of that information via ________ _________.

Answer 7

1 - gene

2 - protein synthesis

Question 8

What is the central dogma of molecular biology?

Answer 8

DNA –> mRNA –> protein

Question 9

mRNAs serve a central function in what?

Answer 9

Transport, regulation and translation of information from DNA nucleotides to a sequence of aa’s that ultimately form fully functional proteins.

Question 10

mRNA expression is controlled by a number of regulatory proteins to regulate many events that affect the cell cycle such as?

Answer 10

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

Question 11

In this lab, we will be investigating which technique?

Answer 11

RNAi

Question 12

The ____ process of RNAi was originally discovered in ______, where it was originally described as what?

Answer 12

1 - in-vivo
2 - plants
3 - post-transcriptional gene silencing

Question 13

RNAi is a powerful tool used to what?

Answer 13

Artificially down-regulate the expression of specific target genes.

Question 14

RNAi is a natural process. How so?

Answer 14

Natural process triggered by dsRNA precursors that are present during viral infection of mammalian systems.

Question 15

Precursors are processed into what, with a length of what? (dsRNA precursors from viral infection)

Answer 15

Short RNA duplexes of 21-28 nucleotides in length.

Question 16

siRNAs consist of _____ bp duplexes, containing both the ____ and ____-_____ strands with ___ ______ that overhang at the __ end.

Answer 16

1 - 19-21
2 - sense and anti-sense
3 - two nucleotides
4 - 3’

Question 17

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.

Answer 17

1 - transfection reagent
2 - polyamines
3 - cytotoxic

Question 18

What do siRNA molecules do upon entering the cell?

Answer 18

Bind to a nuclease complex to form the RISC complex.

Question 19

The RISC complex is ___-dependent.

Answer 19

ATP

Question 20

Following activation of the RISC complex, the siRNA molecule _______.

Answer 20

unwinds

Question 21

The conjoined siRNA molecule and nuclease complex targets the _________ transcript (i.e. __________ ___ strand) by base pair matching.

Answer 21

1 - homologous

2 - complementary RNA

Question 22

This affinity to complementary mRNA allows for the ____-specific ________ of targeted _____ molecules by the RISC complex.

Answer 22

1 - gene
2 - splicing
3 - mRNA

Question 23

The mRNA is cleaved approximately _____ base pairs from the ___ end of the siRNA molecule, thereby causing a reduction in _____.

Answer 23

1 - 12-15
2 - 3’
3 - gene expression levels

Question 24

What is the first task in this lab?

Answer 24

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.

Question 25

What are the factors that must be considered in order to obtain the expected results?

Answer 25

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

Question 26

What has been done to reduce the possibility of RNase contamination?

Answer 26

All solutions, glassware, and plastic-ware will be pre-treated to denature/destroy ribonucleases.

Question 27

What are the solutions pre-treated with?

Answer 27

DEPC which inactivates ribonucleases.

Question 28

What buffers cannot be treated wit DEPC?

Answer 28

Tris based solutions.

Question 29

The ______ is unable to destroy RNases so, glassware will be?

Answer 29

1 - autoclave

2 - baked at 400C for at least 4 hrs

Question 30

Tips and tubes are certified _____ free.

Answer 30

RNase

Question 31

Why are gloves so important?

Answer 31

Major source of RNase.

Question 32

What will we be using to isolate RNA from 293 cells?

Answer 32

RNease Mini kit that is produced by QIAGEN.

Question 33

Following RNA isolation, we will generate ____, and perform _____ to determine the expression levels of ______ and ______ from the siRNA treated cells.

Answer 33

1 - cDNA
2 - pcr
3 - GAPDH and TALIN