ptactical chorinic myeloid leukemia of patients

Aims

To give hands on experience in practical application of RT-qPCR for quantification of mRNA transcripts.

To put into context, and aid revision, for module lectures

  • Lecture: Chromosome cytogenetics analysis
  • Lecture: qPCR
  • Lecture: DNA repair
  • Lecture: Cell singling

Introduction: Chronic myeloid leukemia (CML)

CML is a ‘blood cancer’ (leukemia) characterised by the presences of ‘The Philadelphia (Ph) chromosome’. The Ph chromosome is observed in ~90% of patients with CML and is created by a t(9;22)(q34;q11) chromosome translocation event. The translocation creates a version of a ‘fusion gene’ consisting of sections of BCR (serine/threonine) kinase gene and the ABL (tyrosine) kinase gene (Figure 1).

Expression of BCR/ABL (oncogene) growth signals (Figure 2).

‘Unregulated’ BCR/ABL protein kinase signals ‘drive’ development of CML (Figure 3).

Treatments include the use of TKI inhibitors – to downregulate BCR/ABL activity (Figure 4).

Clinical monitoring of TKI treatment 

The RT-qPCR BCR-ABL1 monitoring test

BCR-ABL1 transcripts are used as a marker for the presence and amount of transcriptionally active Philadelphia-chromosome positive leukemia cells in chronic myeloid leukemia patients (Baccarani et al., J Clin Oncol 2009). The clinically approved and standardised BCR-ABL test is used for quantitatively measuring the blood level of BCR-ABL1 fusion transcript during the course of TKI treatment.

RT-qPCR Test Method

Stage 1: Total RNA is extracted and purified from patient leukocytes.

Stage 2: Total RNA it is then reverse transcribed (RT) to cDNA product

Stage 3: BCR/ABL transcript levels are then quantified by fluorescent real-time quantitative polymerase chain reaction (qPCR). The RT-qPCR test detects BCR-ABL1 (primer sequences) and endogenous control (EC)transcripts; to assess the quality and quantity of RNA and to normalize for potential differences between tests.

Stage 4: The result of the test is then expressed as a percent ratio between the BCR-ABL1 and EC transcript copy number (100 x BCR-ABL1/EC)

The Practical you will perform:

Introduction

The practical you will perform today will employ many of the principles of the BCR-ABL1 monitoring test. However, you will use the test the assess the effect of Imatinib TKI treatment on the K562 CML cancer cell line. The stages up to the RT-qPCR analysis have been performed for you:

Stage 1: Total RNA was extracted and purified from 2 set of K562 cells using the Qiagen Plus RNAeasy kit.

  • Sample 1 cells (untreated) were grown for 48hrs in standard growth media for the K562 cell line (RPMI).
  • Sample 2 cells (treated) were grown in RPMI media supplemented with the BCR/ABl TKI inhibitor – imatinib, at a final concentration of 1 µM.
  • Extracted RNA was tested for quality and quantity using the BioRad Experion microfluidic system(RQI ?7.2 for both samples): See MIQE Guidelines

Stage 2: 1.6µg of total RNA from each sample was then reverse transcribed (RT) to cDNA product using the BioRad iScript Advanced cDNA synthesis kit

Stage 3: You will use fluorescent real-time quantitative polymerase chain reaction (RT-qPCR) to detect the level of BCR-ABL1 and endogenous control transcripts (GUSB) in Sample 1 and Sample 2. The qPCR will be performed using SYBR green analysis using the BioRad Sso Advanced SYBR green qPCR mix not hydrolysis probes as used in the clinical test

You will perform the protocol in 2 parts (see below). 

Stage 4: Finally, using your data and the class data you will determine the normalised expression ratio of BCR-ABL1 in sample 1 and 2. The level of TKI treatment ‘inhibition’ will then be derived using the BioRad CFX manager analysis system.

Note. To perform accurate and precise qPCR analysis, good pipetting skills are essential:

Review Practical: Pipetting, a general introduction

Part 1 – qPCR detection of normalised level of BCR/ABL transcripts in untreated samples

  1. Pipette exactly 30 µL of SYBR green qPCR mix (green tube)into the clear tube containing the cDNA derived from untreated K562 cells
  2. Carefully mix by gently pipetting up and down 4-5 times. Take care not to form any bubbles
  3. Pipette exactly 19 µL of the SYBR green/cDNA ‘mix’ into tube 3 of the white PCR tube strip, and then add 1µL of EC primer set
  4. Pipette exactly 2µL of BA primer set BAinto tube 1 of the white PCR tube strip
  5. Pipette exactly 38 µL of the SYBR green/cDNA ‘mix’ into tube 1, carefully mix by gently pipetting up and down 4-5 times
  6. Transfer exactly 20 µL of the ‘mix’ from tube 1 into tube 2
  7. Carefully place a lid onto the tube strip and bring your set of reactions to the demonstrator (keep reactions on ice)
  8. As a class we will then run all the samples using the BioRad MiniOpticon real-time qPCR thermal cycler

Part 2 – qPCR detection of normalised level of BCR/ABL transcripts in 48hr TKI treated samples. 

  1. Pipette exactly 30 µL of SYBR green qPCR mix (green tube)into the red tube containing cDNA derived from TKI treated K562 cells
  2. Carefully mix by gently pipetting up and down 4-5 times. Take care not to form any bubbles
  3. Pipette exactly 19 µL of the SYBR green/cDNA ‘mix’ into tube 3 of the white PCR tube strip, and then add 1µL of EC primer set
  4. Pipette exactly 2µL of BA primer set BAinto tube 1 of the white PCR tube strip
  5. Pipette exactly 38 µL of the SYBR green/cDNA ‘mix’ into tube 1, carefully mix by gently pipetting up and down 4-5 times
  6. Transfer exactly 20 µL of the ‘mix’ from tube 1 into tube 2
  7. Carefully place a lid onto the tube strip and bring your set of reactions to the demonstrator (keep reactions on ice)
  8. As a class we will then run all the samples using the BioRad MiniOpticon real-time qPCR thermal cycler

Assessment Guidelines

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