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Dong-Er Zhang

Ming Yan

Anita Boyapati

An alternatively spliced isoform of t\(8;21) transcript promotes leukemogenesis

Diverse transcripts are generated by chromosomal translocation 8;21 \(1-6). We reported that one previously unidentified and alternatively spliced transcript, AML1-ETO including ETO exon 9a \(AE9a) \(Figure 1, ref. 1), is leukemogenic in mice and is widely detected in t\(8;21) AML patients \(7). Here, we describe the methods to identify and quantitate the specific A/E9a transcript in t\(8;21) patients samples relative to the AML1-ETO transcript encoding the well known full-length 752 amino acid AML1-ETO protein \(AE).

Reagents:


BM or blood cells from t\(8;21) and non-t\(8;21) leukemia patients obtained with proper consent


RNA isolation reagents: RNeasy mini kit \(Qiagen).


ThermoScriptTM RT-PCR system \(Invitrogen).


FailSafeTM PCR system \(Epicentre Biotechnologies)


Fluorescent Probes \(IDT), 2 &#xB5;M stock


Forward Primer \(IDT), 10 &#xB5;M stock


Reverse Primer \(IDT), 10 &#xB5;M stock


IQ Reaction Supermix \(BioRad) 


Nuclease Free dH20


Real-Time PCR plate \(USA Scientific Template 1 PCR plate 1402-9596)


Template RT Optical Film \(USA Scientific, 2978-2100)


Filter Pipet Tips for PCR





Primers:


First round PCR for AML1-ETO:


AML1 \(RUNX1) exon 4/5 boundary sense primer: 5'-GAGGGAAAAGCTTCACTCTG -3'


ETO \(RUNX1T1, MTG8) exon 9 antisense primer: 5'-CTGTGAAGGAATTCCCGATG-3'.





Second round real time PCR for AE quantitation:


ETO exon 8 sense primer: 5&#x2019;-AGAGTCCCGTCAACCCAGAC-3&#x2019; 


ETO exon 9 antisense primer: 5&#x2019;-ATCTCCTCTGGCACGTATCC-3&#x2019;


probe HEX-CAGTTGCACTAGACGCGCATC-IABLACK \(located across the border of exon 8 and exon 9).





Second round real time PCR for AE9a quantitation:


ETO exon 9a sense primer: 5&#x2019;-TGAGCATTGCTGTCCTGGGTCATA-3&#x2019;


ETO exon 9 antisense primer: 5&#x2019;-TTGGATACTAGATACTGCAAGGGCCG-3&#x2019;ETO 


Exon 9a probe: FAM-TGAGGTCACATTGCTTCTCCAAAGGC-TAM





Software:


For primer and probe design: we used PrimerQuest software available at idtdna.com and Beacon designer 2.0 \(Biosoft International)


Icycler iQ software \(BioRad)

Real-Time Thermocycler \(BioRad iCycler).                                                  Thermocycler \(Whatman Biometra)

**RNA preparation and RT-PCR:**





1. Purify total RNA using the RNeasy mini kit \(Qiagen) from the patient samples \(numbers of cells are variable) according to the manufacturer&#x2019;s protocol. DNase treatment can be performed to eliminate the DNA contamination. 
 


2. cDNA synthesis: Perform the reverse transcription reaction in a 20 &#xB5;l reaction volume assembled in a thin-walled 0.2 ml PCR tube containing:
 1 &#xB5;g of total RNA


1 &#xB5;l Oligo \(dT)20 primer \(50 &#xB5;M)


2 &#xB5;l 10 mM dNTP


add DEPC-treated dH2O to 12 &#xB5;l 


incubate at 65 &#xB0;C for 5 min for denature and place on ice.


Add following mix to each tube


4 &#xB5;l 5x cDNA synthesis buffer


1 &#xB5;l 0.1m DTT


1 &#xB5;l Rnase OUT


1 &#xB5;l DEPC-treated dH2O


1 &#xB5;l ThermoScript RT


Transfer the tubes to the PCR machine and incubate at 55 &#xB0;C for 1 hour and terminate the reaction at 85 &#xB0;C for 5 minutes. 





3. To distinguish AML1-ETO fusion transcripts containing ETO exon 9 or exon 9a from endogenous ETO transcripts, perform PCR to only amplify the fusion transcripts: 
 With 1 &#xB5;l of cDNA template and AML1 exon 4/5 boundary sense primer and ETO exon 9 antisense primer, perform PCR amplifications with the FailSafeTM PCR system \(Epicentre Biotechnologies) for 40 cycles in a volume of 50 &#xB5;l as follows: denaturating 45 s at 94 &#xB0;C, annealing 45 s at 58 &#xB0;C, and extension 90 s at 72 &#xB0;C.





**Relative quantitation of the AE9a and the AE transcripts:**


1. Dilute PCR products from step 3 1:30 in nuclease free dH20. For relative quantitation of AE9a or AE transcripts, real-time PCR should be performed with fluorescent probes specific for either exon 9 or exon 9a of ETO. 
 To assemble 20 &#xB5;l reactions prepare the following mastermix: pipet 10 &#xB5;l of 2x iQ Supermix with sense and antisense primers specific for either exon 9 or exon 9a \(see reagents). In a separate mastermix, add 1 &#xB5;l 30x diluted PCR products from step 3 above as template and H20. Add the fluorescent probe last since it is light sensitive. 





2. Seal the plate with optical film and place in a 96-well thermocycler.
 Program the thermocycler as follows: 95 &#xBA;C 2&#x2019;, 95 &#xBA;C 1&#x2019; and 55 &#xBA;C 25 cycles, 4 &#xBA;C hold.





3. Check for product amplification on a 1% agarose gel and perform quantification with the BioRad iQ software. 
 


4. To confirm and correct for the relative efficiency of probe and primer pairs for exon 9 and exon 9a transcripts, perform a standard curve using serial dilutions of MigR1-A/E or MigR1-A/E9aP DNA as the template.

1. Miyoshi, H. _et al._ t\(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1. _Proc. Natl. Acad. Sci. U. S. A_ **88**, 10431-10434 \(1991).
  2. Nisson, P. E., Watkins, P. C. &amp; Sacchi, N. Transcriptionally active chimeric gene derived from the fusion of the AML1 gene and a novel gene on chromosome 8 in t\(8;21) leukemic cells. _Cancer Genet Cytogenet._ **63**, 81-88 \(1992).
  3. Erickson, P. _et al._ Identification of breakpoints in t\(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt. _Blood_ **80**, 1825-1831 \(1992).
  4. Kozu, T. _et al._ Junctions of the AML1/MTG8\(ETO) fusion are constant in t\(8;21) acute myeloid leukemia detected by reverse transcription polymerase chain reaction. _Blood_ **82**, 1270-1276 \(1993).
  5. Tighe, J. E. &amp; Calabi, F. Alternative, out-of-frame runt/MTG8 transcripts are encoded by the derivative \(8) chromosome in the t\(8;21) of acute myeloid leukemia M2. _Blood_ **84**, 2115-2121 \(1994).
  6. Era, T. _et al._ Expression of AML1 and ETO Transcripts in hematopoietic cells. _Leukemia_ **9 Suppl 1**, S26-S28 \(1995).
  7. Yan, M. _et al._ An alternatively spliced isoform of t\(8;21) transcript promotes leukemogenesis. _Nat. Med._ Published online: 30 July 2006; doi:10.1038/nm1443 \(2006).

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Method Article

Dong-Er Zhang, Ming Yan, Anita Boyapati

Dong-Er Zhang

The Scripps Research Institute

Ming Yan

The Scripps Research Institute

Anita Boyapati

The Scripps Research Institute

DOI:

10.1038/nprot.2006.193

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A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis

by Yan, M. et al.

Nature (17 July, 2006)

An open repository of community-contributed protocols sponsored by Nature Research.

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Dong-Er Zhang, Ming Yan, Anita Boyapati

Dong-Er Zhang

The Scripps Research Institute

Ming Yan

The Scripps Research Institute

Anita Boyapati

The Scripps Research Institute

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10.1038/nprot.2006.193

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A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis

by Yan, M. et al.

Nature (17 July, 2006)

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