Biotechnology siRNA RT-PCR Lab Marking Scheme ( /20) Introduction – 3 marks Materials and Methods As per lab manual (List any protocol changes in this section) Results – 12 marks (total) include legends and labels/titles for all tables, graphs and gel images Written component – 2 marks Do not repeat lab manual methods/protocols Should have a brief paragraph as intro into each result section RNA Isolation - 1 mark Type of sample you received (siNEG, siPTEN, siGAPDH or untransfected cells) Table containing RNA concentration (ng/μl) and purity results (260, 280, 260/280) First strand synthesis, RT-PCR and gel images – properly labelled (4 images total) – 2 marks Sample calculation for RNA required for reverse transcription (μl) each gel image properly labelled – includes lane assignment, # of PCR cycles, band sizes on the 1kb ladder, etc

Question

Biotechnology siRNA RT-PCR Lab Marking Scheme ( /20)

Introduction – 3 marks

Materials and Methods

As per lab manual (List any protocol changes in this section)

Results – 12 marks (total)

**include legends and labels/titles for all tables, graphs and gel images**

Written component – 2 marks

Do not repeat lab manual methods/protocols
Should have a brief paragraph as intro into each result section

RNA Isolation - 1 mark

Type of sample you received (siNEG, siPTEN, siGAPDH or untransfected cells)
Table containing RNA concentration (ng/μl) and purity results (260, 280, 260/280)

First strand synthesis, RT-PCR and gel images – properly labelled (4 images total) – 2 marks

Sample calculation for RNA required for reverse transcription (μl)
each gel image properly labelled – includes lane assignment, # of PCR cycles, band sizes on the 1kb ladder, etc. and/or posted representative data set (for each siNEG, siPTEN, siGAPDH and untransfected cells with all cycles and both primer sets)
What should be labeled for the gel: DNA ladder (mark some of the bands, not all of them if it doesn’t fit), GAPDH and PTEN Primers (which side is which), arrow pointing to size of PTEN and GAPDH bands

Graphs (4 in total) of amplification – based on the densitometry analysis (ImageJ values for amplified DNA bands (y-axis) vs. PCR cycles (x-axis) – 1 mark

Compare your data (from the class, mention which one is actually your group’s result) to posted data set (0.25 marks each)
Analyze all experimental data obtained during your lab section (0.25 marks each) and include graphs for all of them
Include both sets of primers on the same graph (GAPDH primers and PTEN primers)

Table of excel densitometry values you generated using ImageJ software for all PCR cycles and both primer sets and all 4 samples (siNEG, siGAPDH, siPTEN and untransfected) or if only partial results were obtained, analyze what is available or analyze representative data – 0.25 mark each

Sample calculations Required – 2 marks

ONE sample calculation for determining the normalization factor for ONE PCR CYCLE (this can be for NFsiPTEN or NFsiGAPDH)

NFsiPTEN= si(-)_GAPDH primers/si(-)_PTEN primers
NFsiGAPDH = si(-)_PTEN primers/si(-)_GAPDH primers

ONE sample calculations for relative measure of expression (GAPDH or PTEN) due to siRNA for ONE PCR cycle for YOUR experimental results

Equations for determining the relative expression for:

siGAPDH = siGAPDH_GAPDH primers/siGAPDH_PTEN primers x NFsiGAPDH si PTEN = siPTEN_PTEN primers/siPTEN_GAPDH primers x NFsiPTEN

Table summarizing the expression levels for ALL PCR cycles for siGAPDH and siPTEN from for all the cycles for YOUR experimental sample (NOTE: if you were given siNEG, you should also include the relative measure of expression for both siGAPDH and siPTEN using your experimental NFs from your siNEG samples) - 1 mark

Discussion – 5 marks

Conclusion and final remarks is part of this section
Comment on RNA isolation and purity (compare to ‘good’ values)
Comment on graph trends (i.e. should see gradual increase in amplification with increasing PCR cycles for the negative control and lower with siRNA treatment)
State results from experimental gel image (If your experiment did not work provide scientific explanation as to why – ‘human error’ is not a feasible explanation)
Compare results to posted data sets and describe the effect of siRNA treatment (need to state actual calculated values here)
Brief explanation why a normalization factor had to be used and that we used GAPDH for this (also comment on why the determination for GAPDH levels are not accurately reliable)
How these results could be better
Controls, sources of error, replicate experiments etc.