RNA Techniques

Question 1

gene expression (mRNA) in cells can be tested using

Answer 1

1. northern blotting
2. reverse-transcriptase-polymerase chain reaction (RT-PCR)
3. Fluorescent In Situ (in its original place) Hybridization - FISH

Question 2

global expression patterns

Answer 2

• transcriptomics
• provides quantitative (quantity) info about population of RNA species in cells and tissues
  4. DNA microarrays (for RNA NOT DNA)
  5. RNA-sequencing

Question 3

northern blotting

Answer 3

combines gel electrophoresis of RNA w/ nucleic acid hybridization
- less steps than southern blotting
- no denaturing because RNA is single stranded
- no alkaline solution
- used for detection and/or semi-quantification of specific transcripts
steps: RNA extraction from cell –> electrophoresis –> RNA separated by size –> Northern blotting (transfer of RNA to membrane) –> RNA fixed to membrane with UV or heat –> membrane hybridized with labeled probes –> visualization of labeled RNA on X-ray film

Question 4

does southern blotting or northern blotting use an alkaline solution when blotting?

Answer 4

southern blotting (DNA)

Question 5

reverse transcriptase-polymerase chain reaction (RT-PCR)

Answer 5

PCR for RNA –> need double stranded molecule for PCR so we make cDNA
- quick and sensitive technique for mRNA detection and quantification
- reverse transcriptase added to mRNA to make cDNA, which serves as template for PCR amplification of gene of interest –> then quantify by gel electrophoresis
- basic summary: reverse transcribe RNA to cDNA then run normal PCR then gel electrophoresis

Question 6

quantitative reverse transcription PCR (qRT-PCR)

Answer 6

for mRNA detection and quantification
1. denaturation
2. probe hybridization
3. extension/probe hydrolyzation (break bond)
4. fluorescence emission (probe glows)
principle: SYBR Green dye preferentially binds to double stranded DNA –> higher amounts of template DNA = fluorescence more detectable at fewer PCR cycles
- fast + more quantitative

Question 7

Fluorescent In Situ Hybridization (FISH)

Answer 7

tell you location of specific mRNA
- uses fluorescent dyes attached to probes to identify location of specific mRNAs in place in intact tissues or organisms

Question 8

global gene expression analysis

Answer 8

transcriptomics/study of transcriptome: set of all RNA molecules in one or population of cells
- techniques to determine expression pattern: DNA microarrays and RNA sequencing

Question 9

DNA microarray**

Answer 9

slide 23 and 26 = good diagrams
template for DNA microarrays is mRNA –> that’s what makes it an RNA technique
- microarray chips represent gene sequences from all genes in organisms genome
- converting template mRNA to cDNA and using that cDNA for comparison to experimental sample
- helpful because can measure thousands of genes simultaneously
1. mRNA extraction
2. reverse transcribe mRNA to cDNA –> fluorescent labeling for control and experimental samples
3. fluorescently labeled cDNA hybridized with gene chip and see if it binds –> comparison method: control sample (cDNA) mixed with experimental sample (e.g. cancer cell) –> each group fluorescently labeled –> any time fluorescent probe from either group shows up means complement was found on gene chip = within these cells, mRNA produced for that gene

Question 10

novel digital barcode technology for direct precise measurement of mRNA analytes is called _________

Answer 10

nanostring technology

Question 11

limitations of mRNA measurements in gene expression analysis

Answer 11

applies to DNA microarrays and RNA sequencing
1. may not reflect protein levels
2. may not reveal the form of protein translated from mRNA (if post-translationally modified, etc.)
3. will not reflect protein activity