Group 2 QPCR results

Group 2 QPCR results

Run 2

BIOL2267 Functional Genomics & Proteomics

Quantitative PCR (real-time PCR)

Introduction

You have been hired as a medical research assistant in our lab to investigate medicinal plants to augment cancer development. In this practical, you will use molecular biology to quantify the effect of an exotic herb that reduces melanoma on human cells using QPCR. This herb acts on the Nuclear factor kappa-light-chain-enhancer of activated Bcells (NF-kB) pathway that causes downstream activation of inflammatory and antioxidant derivatives including Tumour necrosis factor alpha (TNF-), Nuclear factor erythroid 2-related factor 2 (Nrf2) compared to the housekeep gene GAPDH (Figure 1).

3175158115Herb concentrations (mg/mL)

0, 0.5, 1.0, 2.0, 3.0, 4.0

00Herb concentrations (mg/mL)

0, 0.5, 1.0, 2.0, 3.0, 4.0

373316523495Melanoma cells tested: C1, C32, D24

Control cell: HaCat

00Melanoma cells tested: C1, C32, D24

Control cell: HaCat

Figure 1. Signalling pathways by active phytochemicals to augment cancer chemotherapy and immunotherapy (https://www.frontiersin.org/articles/10.3389/fonc.2022.834072/full). TLRs=Toll-like receptors.

Learning outcomes

To accurately and consistently dispense small volumes with a micropipette.

To create a standard curve for amplification efficiency for qPCR.

To perform qPCR on untreated and treated samples.

To analyse qPCR performance, melt curves and co-efficiency cohorts.

To write a scientific report on the benefits of our exotic herb for chemotherapy.

Instructions

A. Calculation of cDNA to use

cDNA were extracted and diluted to 1 g/L for you to use.

Work in groups and set up the experiment as a class. The Gene of Interest (GOI) primers change with stimulation and treatment (=NFkB, TNFa, Nrf, p53). Housekeeping (HK) primers are constitutively expressed, always on and shouldnt change (=GAPDH).

You will form 2 groups.

Group 1 will setup the standard curve with different primers xng cDNA and controls

Group 2 will setup the cDNA samples with different primers and 1ng cDNA.

-23558540640000B. Setup of the standard curve this has already been done by group 1

Group 1 (8 students) will set up the standard curve using the 4 different primers with different cDNA concentrations of untreated H cells (labelled as HU). Include a no template control (NTC=0.0ng cDNA). Refer to table 1.

Table 1. Setup of the standard curve and controls.

Student Well 1 Well 2 Well 3 Well 4

A Standard GAPDH

0ng HU Standard GAPDH

1ng HU Standard GAPDH

2ng HU Standard GAPDH 4ng HU

B Standard GAPDH

0ng HU Standard GAPDH

1ng HU Standard GAPDH

2ng HU Standard GAPDH 4ng HU

C Standard NFkB

0ng HU Standard NFkB

1ng HU Standard NFkB

2ng HU Standard NFkB

4ng HU

D Standard NFkB

0ng HU Standard NFkB

1ng HU Standard NFkB

2ng HU Standard NFkB

4ng HU

E Standard TNFa

0ng HU Standard TNFa

1ng HU Standard TNFa

2ng HU Standard TNFa

4ng HU

F Standard TNFa

0ng HU Standard TNFa

1ng HU Standard TNFa

2ng HU Standard TNFa

4ng HU

G Standard Nrf

0ng HU Standard Nrf

1ng HU Standard Nrf

2ng HU Standard Nrf

4ng HU

H Standard Nrf

0ng HU Standard Nrf

1ng HU Standard Nrf

2ng HU Standard Nrf

4ng HU

Calculate what will be needed to create your mastermix (table 2). Why do you need a mastermix? Make your mastermix containing your qPCR mix, primers (forward and reverse) multiplied by 5 (fill in table 2). Double check your calculations! Remember you need enough for 4 samples so there should be plenty left over! Too little or too much and what happens?

Table 2. Constituents for a qPCR standards (Sensimix Sybr no rox).

Component (label) L per reaction [Final] Mastermix for standards

L (x5)

qPCR mix (Q) 32512038735006 1x Forward Primer (gene F) 49974523495mastermix

020000mastermix

1 500nM Reverse Primer (gene R) 1 500nM DEPC water * - * (add separate)

cDNA (HU) (?ng) * - * (add separate)

Total l aliquoted per well 8 - Total l volume 12.5 - *indicates variable amounts required to make the final total volume

Wearing gloves, wipe down your bench area with 70% ethanol.

Make the standards mastermix according to table 2 containing the qPCR mix and primers (forward and reverse) in an eppendorf. Mix by pipetting up and down gently.

5080-70739000Grab a QPCR 4-well. Make note of where well 1 is by drawing a dot on the lid.

Transfer 8uL of the mastermix to each well.

If you get to the last well and there is not enough, you have over pipetted and its game over!

Then add cDNA to the concentration required. Fill up to total volume with the DEPC water.

Close the lids. Remove air bubbles by flicking or tapping wells. Keep on ice and in darkness.

Your sample is now ready to run in a qPCR and you have finished the practical.

Wait for the rest of the class to load their PCR strip tubes. Note your group number and details of each person who pipetted what to write in your report.

C. Setup of the cDNA samples.

Group 2 will have 2 students in each group.

Each group will set up the wells according to table 3 exactly.

Table 3. Setup of the cDNA samples with and without 1mg/mL plant extract treatment (untreated=U, treated=T) for each of the different cell types (melanomas: C1, C32, D24, control H). Different primers are H

Group Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8

C1 cells

A Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA treated Sample GAPDH

1ng cDNA

treated Sample GAPDH

1ng cDNA

treated B Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA treated Sample NFkB

1ng cDNA

treated Sample NFkB

1ng cDNA

treated C Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

treated Sample TNFa

1ng cDNA

treated Sample TNFa

1ng cDNA

treated D Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

treated Sample Nrf

1ng cDNA

treated Sample Nrf

1ng cDNA

treated Group Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8

C32 cells

E Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA treated Sample GAPDH

1ng cDNA

treated Sample GAPDH

1ng cDNA

treated F Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA treated Sample NFkB

1ng cDNA

treated Sample NFkB

1ng cDNA

treated G Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

treated Sample TNFa

1ng cDNA

treated Sample TNFa

1ng cDNA

treated H Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

treated Sample Nrf

1ng cDNA

treated Sample Nrf

1ng cDNA

treated

Group Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8

D24 cells

I Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA treated Sample GAPDH

1ng cDNA

treated Sample GAPDH

1ng cDNA

treated J Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA treated Sample NFkB

1ng cDNA

treated Sample NFkB

1ng cDNA

treated K Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

treated Sample TNFa

1ng cDNA

treated Sample TNFa

1ng cDNA

treated L Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

treated Sample Nrf

1ng cDNA

treated Sample Nrf

1ng cDNA

treated Group Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8

H cells

M Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA untreated Sample GAPDH

1ng cDNA treated Sample GAPDH

1ng cDNA

treated Sample GAPDH

1ng cDNA

treated N Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA untreated Sample NFkB

1ng cDNA treated Sample NFkB

1ng cDNA

treated Sample NFkB

1ng cDNA

treated O Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

untreated Sample TNFa

1ng cDNA

treated Sample TNFa

1ng cDNA

treated Sample TNFa

1ng cDNA

treated P Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

untreated Sample Nrf

1ng cDNA

treated Sample Nrf

1ng cDNA

treated Sample Nrf

1ng cDNA

treated Create a mastermix. Calculate what will be needed to create your mastermix as a group for 6 samples (table 4). Why do you need to make calculations for 7 samples? Check your calculations.

Table 4. Constituents for a qPCR reactions (Sensimix Sybr no rox kit).

Component (label) L per reaction [Final] Mastermix for cDNA samples

L (x7)

qPCR mix (Q) 4013203810006 1x Forward Primer (PrimerF) 50673074930mastermix

020000mastermix

1 500nM Reverse Primer (PrimerR) 1 500nM DEPC water 3.5 - cDNA (1ng) (C1U/T, C32U/T, D24U/T, HU/T) 1 - Add last (add separately)

Total l aliquoted per well 11.5 - Total l volume 12.5 - Wearing gloves, wipe down your bench area with 70% ethanol.

Add the qPCR mix, primers (forward and reverse), and water to an eppendorf to create a mastermix according to table 4. Mix by pipetting up and down gently.

Grab a qPCR 4-well. Do NOT draw on it.

Aliquot 11.5uL of the mastermix to wells according to table 4. If you get to the last well and there is not enough, you have over pipetted!

Add 1 L DNA to each well and gently mix with your pipette without creating air bubbles.

Close the lids. Remove air bubbles by flicking or tapping wells. Keep on ice and in darkness.

Bring it to your demonstrator to label your tube. Make sure you tell us what the cell and gene is. Note your group number and details of each person who pipetted what to write in your report.

Your sample is now ready to run in a qPCR and you have finished the practical.

Practical report - Results will be available online

Results containing the below will be available online so keep an eye out for it :

A standard curve graph contains the correlation coefficient values with the slope and R2 value for all your primers.

A data graph contains Ct values with adjusted threshold line for your cDNA samples.

A melt curve graph for your cDNA samples that contains peaks about amplification and the double template dissociation.

There will be a tutorial where you can ask questions about your report. Attempt to calculate the primer efficiency and gene expression BEFORE the tutorial so you can maximise your learning.

Write up this practical analysis to answer the rubric below. Refer to the marking scheme below to get the best grade.

Each student must submit online via the assignment link by the due date.

Things to include in your report:

How did you do (results, accuracy) and what can you improve?

How did the class do (results, accuracy) and what can be done to improve?

Efficiency of primers (GOI and HK)

Gene expression untreated for each cell line (include calculations)

Gene expression treated with the herb for each cell line (include calculations)

Summary graph of gene expression

What was the best treatment?

Is the herb a good product for chemotherapy?

Rubric Full marks Half marks No marks

Results and writing (10)

Results are well presented. Communication is succinct and concise using appropriate terminology and explains well the outcomes. Results are partially presented. Communication is partially coherent with some appropriate terminology and partially explains the outcomes. Fails to demonstrate pass standards, or communication is so poor as to make assessment on this criterion impossible.

Analysis (10)

Answers all questions and analysis shows critical thinking and depth of knowledge.

Answers some questions or analysis shows some critical thinking with some depth of knowledge. Fails to demonstrate pass standards, or communication is so poor as to make assessment on this criterion impossible.

Total (20)