Measuring the Transcriptome

Genome

the entire compliment of DNA sequence within the cells of an organism

Transcriptome

groups of genes that are expressed within a cell or tissue

• same in all neurones

isolating RNA

• classic trizol method

• 1. cells/tissue are lysed/homogenized with trizol and physical methods (so DNA is spread)

• 2. chloroform added and sample vortexed

• 3. centrifuge for phase separation

• RNA phase (top phase) transferred to another tube and precipitated with ethanol (or other solvent → as long as there are no enzymes that could damage the RNA)

PCR

• polymerase chain reaction

• used to measure gene/RNA expression (QPCR, in situ hybridisation) or manipulate genome/transcriptome (cloning and CRISPR)

• uses Taq polymerase which has been modified to improve yield and efficiency

• three step process:

  • denaturation

  • annealing

  • elongation

• can use hot start to activate enzyme in some cases

Denaturation in PCR

• temperature at 92-94C

• double stranded DNA separates into two single strands

Annealing in PCR

• temperature at 50-70C (primer dependent)

• primer ( 20-24bp) binds to its complimentary sequence in DNA to be amplified

• forward primer binds to 5’ and reverse primer binds to 3’

Elongation in PCR

• temperature is around 72C

• DNA polymerase binds to the 3’ end of the primers and copies the complementary sequence

General lab protocol for PCR

• add things to eppendorf:

  • template DNA/RNA

  • primers

  • Taq polymerase

  • Buffer

  • dNTPs

• amplify in thermocycler

• run on agarose gel

• visualise with UV light

Gel electrophoresis

• visualise PCR products by running on agarose gel and imaging under UV light

  • can see if their is a product and estimate its size

Quantitative PCR (QPCR)

• DNA detected with fluorescent probe

• PCR reaction is exponential → a detection threshold (Cycle threshold = CT) is set (higher starting amount = sooner reaches detection threshold)

• can calculate the amount of DNA in samples compared to a control

  • calculate relative expression and normalise it

    • CT increase of 1 = increase by 50% as sample is doubled each cycle

• two types: SYBRGreen and Taqman

SYBRGreen QPCR

• SYBRGreen molecule becomes fluorescent when intercalated with double stranded DNA, so more fluorescence means more double stranded DNA is present

• analysed using melt analysis (measures fluorescence after increasing temp by 1C at a time)

• advantage → uses regular PCR primers

• disadvantage → cannot differentiate between gene of interest and other products

TAQMan QPCR

• has forward, reverse and probe primers

  • probe primers have a fluorescent molecule which fluoresces when digested by Taq

    • more fluorescence = more double stranded DNA has been digested

• advantage → highly specific

• disadvantage → needs special and expensive primers

DNA microarrays

• glass or silicone slide with thousands of microscopic DNA sports]

• allows for relative quantification of many genes at once

• each DNA spot has a known location and different sequence

• samples are fluorescently labelled and incubated with the microarray. These bind to their complimentary DNA spots

• fluorescence is measured → more target gene = more fluorescence

  • relative expression is calculated (there is a control for comparison)

• Different types: Spotted DNA arrays and Affimetix gene chips

Spotted DNA arrays

• type of DNA microarray

• PCR products of known genes spotted on support (glass, plastic or nylon)

• control and treatment samples labelled with fluorescent colours and hybridized to same microarray

• relative changes in gene expression calculated by comparing signals from control and treated

  • must be in the same microarray, so one per each treatment

Affy Gene Chips

• developed by Affymetrix

• short probes on glass slides

• each sample labelled into different gene chip

• relative changes in gene expression calculated by comparing readings of the chip with control and treated samples

  • normalised between chips using affymetrix oligos so only needs one control

• expensive as company design is required

Sanger sequencing

• first generation of sequencing

• makes many products ending with specific base pairs

• electrophoresis in microcapillary tube with smallest moving fastest and furthest

• can read the sequence and spot mutations

• is a slow process as genes are read one at at time

• the normal sequence must be known to spot mutations

Next generation (2nd generation) sequencing

• Library preparation → random fragmentation of DNA and ligation via custom linkers to solid phases (microbit support) within a bubble → these are called libraries

• Amplification → each DNA fragment is amplified (clones stay close to original DNA fragment)

• Sequencing → pyrosequencing or reverse terminator sequencing

  • Pyrosequencing → pyrophosphate released with every base pair

    • this is converted to ATP by ATP sulfurylase and adenosine

    • ATP is used to convert luciferin to oxyluciferin

    • oxyluciferin produces light detectable with a camera, with each flash telling the next base pair

  • Reverse terminator sequencing (Illumia)

    • the same as sanger sequencing but uses reversible termination