Real-time PCR Training

The abbreviation of Real-Time PCR is

• PCR

• qPCR

• RT-PCR

• RT-qPCR

Polymerase Chain Reaction (PCR) allows scientists to amplify (make many copies of) a small amount of DNA using a repetitive process involving DNA polymerase and short DNA primers

• Yes

• No

Absolute Quantification Experiment:

• Requires probe assay that consists of two allele-specific probes.

• Requires a standard curve to determine the absolute quantity of a

  target in samples.

• Requires endogenous control to establish

  relative expression levels that reflect the real biological changes

Relative Quantification Experiment:

• Requires probe assay that consists of two allele-specific probes.

• Requires a standard curve to determine the absolute quantity of a

  target in samples.

• Requires endogenous control to establish

  relative expression levels that reflect the real biological changes

Genotyping Experiment:

• Requires probe assay that consists of two allele-specific probes.

• Requires a standard curve to determine the absolute quantity of a

  target in samples.

• Requires endogenous control to establish

  relative expression levels that reflect the real biological changes

Threshold Cycle (Ct):

• Is the level of signal that reflects statistically significant increase over the calculated baseline signal.

• Is the cycle number at which the fluorescent signal of the reaction crosses the threshold.

• refers to how well the PCR doubles the

  amount of DNA during each cycle.

• Is ideally close to -3.32.

Below are components of a PCR reaction (choose ALL that apply).

• DNA Template

• DNA Polymerase

• Primers

• Nucleotides

Below are the Cycling steps in a PCR (choose ALL that apply).

• Denaturation

• Annealing

• Extension

• Truncation

Advantage of Real-Time PCR over PCR.

• Involve amplification of DNA sequence

• Can accomodate End-Point analysis

• Can provide Qualitative results

• Can provide Quantitative results

Below are the Chemistries of Real-Time PCR (choose ALL that apply).

• SYBR GREEN

• Probe detection

• Ethidium Bromide (EtBr)

• SYBR Safe