Lab 2 - RNA Interference, RNA Isolation, and Intro to RT-PCR

Question 1

What are the tasks to be completed on day 1?

Answer 1

• RNA isolation
• RNA quantification
• Reverse transcription (cDNA synthesis)

Question 2

What are the tasks to be completed on day 2?

Answer 2

Semi-quantitive RT-PCR

Question 3

What are the tasks to be completed on day 3?

Answer 3

Preparation of agarose gel

Question 4

What are the tasks to be completed on day 4?

Answer 4

Agarose gel electrophoresis

Question 5

What is “total RNA”?

Answer 5

A common term used to describe the entire complement of RNA molecules found in any given cell and includes mRNA, tRNA, and rRNA

Question 6

How is mRNA transcribed?

Answer 6

As a single-stranded molecule containing complementary ribonucleotides from gene-containing template

Question 7

What is tRNA involved in?

Answer 7

Protein synthesis within the ribosome (composed of rRNA)

Question 8

How does tRNA function?

Answer 8

By converting the information carried by mRNA into a corresponding amino acid sequence

Question 9

What do snRNA and miRNA do?

Answer 9

Play a variety of roles in regulatory function

Question 10

What is snRNA involved in?

Answer 10

Maturation of mRNA

Question 11

What is miRNA involved in?

Answer 11

Regulation of gene expression

Question 12

What is mRNA the key link between?

Answer 12

The information stored within a gene and the expression of that information via protein synthesis (Central Dogma)

Question 13

Where does the genetic flow of information from DNA to mRNA to protein occur?

Answer 13

In all cells where the genetic carrier is double stranded DNA

Question 14

What central functions does mRNA serve?

Answer 14

Transport, regulation, and translation of information from DNA nucleotides to a sequence of amino acids that ultimately form fully functional proteins

Question 15

How is mRNA expression controlled?

Answer 15

By a number of regulatory proteins in an effort to manage multiple events that affect the cell cycle (level/stability of mRNA, ability to form functional proteins following post-transcriptional modification, mRNA translocation within the cell)

Question 16

Overall, what is the cellular control of gene expression?

Answer 16

A complex process that involves significant interaction between regulatory and/or elements and multiple RNA molecules

Question 17

What is RNAi (gene silencing) a powerful tool for?

Answer 17

Artificially down-regulate the expression of specific target genes in organisms

Question 18

What is RNAi?

Answer 18

A natural process triggered by double-stranded RNA precursors that are present during viral infection of mammalian systems

Question 19

How do the precursors in RNAi vary?

Answer 19

In length and are processed into short RNA duplexes of 21 to 28 nuclotides in length

Question 20

What are the 21 to 28 nucleotide duplexes responsible for?

Answer 20

Gene silencing by specific mechanisms that vary in different systems (mediation of translational repression, guidance of mRNA degradation, alteration of chromatin structure)

Question 21

What do siRNAs consist of?

Answer 21

19-21 base pair duplexes, containing both sense and anti-sense strands with 2 nucleotides that overhang at the 3’ end

Question 22

What happens when siRNAs are involved in RNAi?

Answer 22

• siRNAs are translocated into mammalian cells
• siRNA molecules bind to a nuclease complex to form an RNA-inducing signaling complex (RISC)
• ATP-dependent RISC complex is activated and the siRNA molecule unwinds
• The conjoined siRNA and nuclease complex targets the homologous transcript (complementary RNA strand)
• Splicing of targeted mRNA molecules by RISC complex

Question 23

How is siRNA translocated into mammalian cells?

Answer 23

Using a transfection reagent containing a combination of polyamines that allow for the transfection of small RNA molecules into the cytoplasm without cytotoxic impacts to the cell

Question 24

How does the conjoined siRNA and nuclease complex target the homologous transcript (complementary RNA strand)?

Answer 24

By base pair matching

Question 25

What allows for the gene-specific splicing of targeted mRNA molecules by the RISC complex?

Answer 25

The affinity of the siRNA and RISC complex to complementary RNA

Question 26

What does the cleavage of mRNA at approximately 12-15 bp from the 3' end of the siRNA molecule cause?

Answer 26

A reduction in cellular gene expression levels

Question 27

What cells are we isolating total RNA from?

Answer 27

Human embryonic kidney (HEK293) cells that have been transfected with either siPTEN or negative control siRNA for 48 hours, or non-transfected cells

Question 28

What needs to be taken into consideration in order to obtain the expected results?

Answer 28

- Chemical structure of RNA (sensitive to hydrolysis and degradation) - Single-stranded nature of RNA ("less protected") - Abundance of RNAse enzymes (can wreak havoc on experiment)

Question 29

How will the possibility of contamination be reduced?

Answer 29

- All solutions, glassware, and plastic-ware will be pre-treated to denature and/or destroy ribonucleases - Solutions used in the isolation of RNA are pre-treated with diethylpyrocarbonate (DEPC), which inactivated ribonucleases - Glasseware will be baked at 400 degrees C for at least 4 hours - Tips and tubes will be certified as "RNAse" and "DNAse" free - Wear gloves

Question 30

During the isolation of RNA from 293 cells, what will we be using?

Answer 30

An RNeasy mini kit that is commercially available from QIAGEN