BIOL340 EXAM QUESTIONS AND ANSWERS WITH COMPLETE
SOLUTIONS GRADED A++
real time pcr/qpcr
amplifies and measures dna in real time using fluorescent markers, only allows relative
quantification to house keeping gene
methods of measuring mrna abundance
in extracts by rnaseq if you dont have prior genetic info, microarrays if you know the
target gene of interest, qrt-pcr (ct and ddpcr), in situ hybridisation for exact location
reverse transcription pcr
rt rna into cdna to be pcred
what quantitative rt-pcr compares to
comparing data to amount of tissue (innaccurate due to wet weight), number of cells
(inaccurate due to degradation), rna content (less degradation), external standard (yas),
house keeping gene (best)
qrt-pcr primer requirements
exon spanning primers will prevent amplification of genomic DNA and only pcr cdna
made from mrna, also low gc content, no hairpins binding 3' end or primer dimers,
should be similar melting temps
qrt-pcr primer options
can be multiplexed, use a bunch of random primers to get everything but will include not
just mrna, oligot primers for just mrna but misses non coding, gene specific
, rnaseq
cdna sequenced, sequences compared to database to identify transcripts, then
quantifies the number of transcripts
gene ontology
molecular function, cellular location, biological process
expression microarrays
specific arrays - oligo capture probes on glass slide, cDNA sample added then
hybridized to array, imaged and fluorescent intensity determined as a metric for
abundance
taqman probes
for qrtpcr, probe anneals to centre of template containing a dye and quencher, when
dna polymerase pcrs it the quencher is gone allowing florescence from the dye - very
specific as only template will fluoresce
interchelating dye
for qrtpcr, interchelates randomly into dna molecule during annealing, increases every
cycle, allows quantification but cant distinguish from contaminant or other dna pieces in
the pcr
controls for qrtpcr
no template control, closed tubes, separate pipettes
MIQE guidelines
ensuring reaction priming efficiency is the same, use multiple house keeping genes
things to consider for qrtpcr
primer design, fluorescent method, controls, miqe guidelines, housekeeping genes
SOLUTIONS GRADED A++
real time pcr/qpcr
amplifies and measures dna in real time using fluorescent markers, only allows relative
quantification to house keeping gene
methods of measuring mrna abundance
in extracts by rnaseq if you dont have prior genetic info, microarrays if you know the
target gene of interest, qrt-pcr (ct and ddpcr), in situ hybridisation for exact location
reverse transcription pcr
rt rna into cdna to be pcred
what quantitative rt-pcr compares to
comparing data to amount of tissue (innaccurate due to wet weight), number of cells
(inaccurate due to degradation), rna content (less degradation), external standard (yas),
house keeping gene (best)
qrt-pcr primer requirements
exon spanning primers will prevent amplification of genomic DNA and only pcr cdna
made from mrna, also low gc content, no hairpins binding 3' end or primer dimers,
should be similar melting temps
qrt-pcr primer options
can be multiplexed, use a bunch of random primers to get everything but will include not
just mrna, oligot primers for just mrna but misses non coding, gene specific
, rnaseq
cdna sequenced, sequences compared to database to identify transcripts, then
quantifies the number of transcripts
gene ontology
molecular function, cellular location, biological process
expression microarrays
specific arrays - oligo capture probes on glass slide, cDNA sample added then
hybridized to array, imaged and fluorescent intensity determined as a metric for
abundance
taqman probes
for qrtpcr, probe anneals to centre of template containing a dye and quencher, when
dna polymerase pcrs it the quencher is gone allowing florescence from the dye - very
specific as only template will fluoresce
interchelating dye
for qrtpcr, interchelates randomly into dna molecule during annealing, increases every
cycle, allows quantification but cant distinguish from contaminant or other dna pieces in
the pcr
controls for qrtpcr
no template control, closed tubes, separate pipettes
MIQE guidelines
ensuring reaction priming efficiency is the same, use multiple house keeping genes
things to consider for qrtpcr
primer design, fluorescent method, controls, miqe guidelines, housekeeping genes
