Lecture 8- Methods for Transcriptome analyses

Question 1

Quantitative PCR

Answer 1

reverse transcribe mRNA
PCR with gene of interest amplification with flurorecent dye added

Fluorecence measured
amount of dye at end is equivalent to starting material

Question 2

how to analysis of Q-PCR data

Answer 2

normalise between samples
different amplification rates- use housekeeping gene- where level of gene expression cDNA is proportional

Compare to a standard curve

Question 3

Advantages and Disadvangtages- QPCR

Answer 3

Accurate and Specific

Question 4

Nanostring

Answer 4

molecular barcodes specfic for gene
added and MB purified
immobilized and imaged using scanner

Question 5

Advantages and Disadvangtages- NanoString

Answer 5

ADV-Easy and quick

DIS- Expensive and only detect a limited amount of genes

Question 6

Type of microarray and how it is produced and designs

Answer 6

Oligonucleotide microarray
photolithography
use of light, filtered through a template to remove specfic caps on array so probes can be printed on

Consider
How many genes
prob no, probs per gene
probe design(3’ end)

Question 7

How do micrarray experiments work

Answer 7

reverse transcribe RNna to cDNA, then invtro transcription with dye, labelled RNA(with dye). Put on slide and left to hybridise, washed and scanned flurecense.

Question 8

Fluorecent sequencing technologies

Answer 8

Cyclic reversible termination
sequence by ligation
single nucleotide addition
Pacific biosciences

Question 9

Electrostatic sequencing technologies

Answer 9

Ion torrent

Nanopore