OPTIMISATION OF RNA EXTRACTION AND RT-QPCR FOR THE QUANTIFICATION OF AID IN ARCHIVED FFPE BIOPSY SAMPLES: ONLY TWO SECTIONS OF 4 MM TISSUE ARE REQUIRED
(Abstract release date: 05/21/15)
EHA Library. Alishlash O. 06/12/15; 102969; PB1947
Disclosure(s): University of LiverpoolDepartment of Clinical and Molecular Cancer Medicine
Omar Alishlash
Contributions
Contributions
Abstract
Abstract: PB1947
Type: Publication Only
Background
Activation induced cytidine deaminase (AID) induces genomic alterations required for immunoglobulin SHM degradation and CSR in normal B cells. The mutagenic effects of AID may also contribute to genomic instability and adverse outcome in B-cell malignancies. We sought to address this question by measuring levels of AID mRNA and protein in formalin fixed paraffin embedded (FFPE) biopsy samples from patients with follicular lymphoma (FL). Since measuring specific mRNAs in FFPE samples is notoriously challenging owing to RNA
Aims
in this study, we aimed to first optimise the conditions for mRNA extraction, cDNA synthesis and qPCR amplification.
Methods
8 FFPE tissue samples stored in a local FL tissue bank for 0.5 – 14 years were used for this optimisation. Using 2 sections of 4μm-thick tissue, we found that a better yield of RNA was obtained with the Qiagen RNeasy FFPE Kit as compared to the Promega ReliaPrep™ FFPE Total RNA Kit (2 μg vs 0.9 μg in average).
Results
The yield was further increased (65 %) by modification of the deparafinization procedure. Using random primers for cDNA synthesis combined with a pair of qPCR primers targeting a 100-bp sequence spanning two exons, we found that AID mRNA was consistently and reproducibly detected in both recently obtained and old FFPE samples. Applying our method to artificially degraded RNA extracted from B-cell line cells, we found that AID mRNA levels were not changed when the RNA integrity number (RIN) varied between 2.1 and 10. Applying this criterion to archived FFPE samples from patients with FL that had been stored for between 0.5 and 5 years allowed us to successfully quantify AID mRNA levels in 33/56 (59%) cases. To validate our findings, levels of AID mRNA were compared to levels of AID protein detected in all of the 33 samples by IHC; a significant positive correlation (P < 0.001) was observed.
Summary
In summary, we have shown that a sufficient quantity of mRNA for RT-qPCR can be extracted from only two sections of 4mm-thick FFPE tissue if an optimised protocol is applied. Our findings are relevant not only to the measurement of AID but to all situations where mRNA quantification is required and tissue availability is a limiting factor.
Session topic: Publication Only
Type: Publication Only
Background
Activation induced cytidine deaminase (AID) induces genomic alterations required for immunoglobulin SHM degradation and CSR in normal B cells. The mutagenic effects of AID may also contribute to genomic instability and adverse outcome in B-cell malignancies. We sought to address this question by measuring levels of AID mRNA and protein in formalin fixed paraffin embedded (FFPE) biopsy samples from patients with follicular lymphoma (FL). Since measuring specific mRNAs in FFPE samples is notoriously challenging owing to RNA
Aims
in this study, we aimed to first optimise the conditions for mRNA extraction, cDNA synthesis and qPCR amplification.
Methods
8 FFPE tissue samples stored in a local FL tissue bank for 0.5 – 14 years were used for this optimisation. Using 2 sections of 4μm-thick tissue, we found that a better yield of RNA was obtained with the Qiagen RNeasy FFPE Kit as compared to the Promega ReliaPrep™ FFPE Total RNA Kit (2 μg vs 0.9 μg in average).
Results
The yield was further increased (65 %) by modification of the deparafinization procedure. Using random primers for cDNA synthesis combined with a pair of qPCR primers targeting a 100-bp sequence spanning two exons, we found that AID mRNA was consistently and reproducibly detected in both recently obtained and old FFPE samples. Applying our method to artificially degraded RNA extracted from B-cell line cells, we found that AID mRNA levels were not changed when the RNA integrity number (RIN) varied between 2.1 and 10. Applying this criterion to archived FFPE samples from patients with FL that had been stored for between 0.5 and 5 years allowed us to successfully quantify AID mRNA levels in 33/56 (59%) cases. To validate our findings, levels of AID mRNA were compared to levels of AID protein detected in all of the 33 samples by IHC; a significant positive correlation (P < 0.001) was observed.
Summary
In summary, we have shown that a sufficient quantity of mRNA for RT-qPCR can be extracted from only two sections of 4mm-thick FFPE tissue if an optimised protocol is applied. Our findings are relevant not only to the measurement of AID but to all situations where mRNA quantification is required and tissue availability is a limiting factor.
Session topic: Publication Only
Abstract: PB1947
Type: Publication Only
Background
Activation induced cytidine deaminase (AID) induces genomic alterations required for immunoglobulin SHM degradation and CSR in normal B cells. The mutagenic effects of AID may also contribute to genomic instability and adverse outcome in B-cell malignancies. We sought to address this question by measuring levels of AID mRNA and protein in formalin fixed paraffin embedded (FFPE) biopsy samples from patients with follicular lymphoma (FL). Since measuring specific mRNAs in FFPE samples is notoriously challenging owing to RNA
Aims
in this study, we aimed to first optimise the conditions for mRNA extraction, cDNA synthesis and qPCR amplification.
Methods
8 FFPE tissue samples stored in a local FL tissue bank for 0.5 – 14 years were used for this optimisation. Using 2 sections of 4μm-thick tissue, we found that a better yield of RNA was obtained with the Qiagen RNeasy FFPE Kit as compared to the Promega ReliaPrep™ FFPE Total RNA Kit (2 μg vs 0.9 μg in average).
Results
The yield was further increased (65 %) by modification of the deparafinization procedure. Using random primers for cDNA synthesis combined with a pair of qPCR primers targeting a 100-bp sequence spanning two exons, we found that AID mRNA was consistently and reproducibly detected in both recently obtained and old FFPE samples. Applying our method to artificially degraded RNA extracted from B-cell line cells, we found that AID mRNA levels were not changed when the RNA integrity number (RIN) varied between 2.1 and 10. Applying this criterion to archived FFPE samples from patients with FL that had been stored for between 0.5 and 5 years allowed us to successfully quantify AID mRNA levels in 33/56 (59%) cases. To validate our findings, levels of AID mRNA were compared to levels of AID protein detected in all of the 33 samples by IHC; a significant positive correlation (P < 0.001) was observed.
Summary
In summary, we have shown that a sufficient quantity of mRNA for RT-qPCR can be extracted from only two sections of 4mm-thick FFPE tissue if an optimised protocol is applied. Our findings are relevant not only to the measurement of AID but to all situations where mRNA quantification is required and tissue availability is a limiting factor.
Session topic: Publication Only
Type: Publication Only
Background
Activation induced cytidine deaminase (AID) induces genomic alterations required for immunoglobulin SHM degradation and CSR in normal B cells. The mutagenic effects of AID may also contribute to genomic instability and adverse outcome in B-cell malignancies. We sought to address this question by measuring levels of AID mRNA and protein in formalin fixed paraffin embedded (FFPE) biopsy samples from patients with follicular lymphoma (FL). Since measuring specific mRNAs in FFPE samples is notoriously challenging owing to RNA
Aims
in this study, we aimed to first optimise the conditions for mRNA extraction, cDNA synthesis and qPCR amplification.
Methods
8 FFPE tissue samples stored in a local FL tissue bank for 0.5 – 14 years were used for this optimisation. Using 2 sections of 4μm-thick tissue, we found that a better yield of RNA was obtained with the Qiagen RNeasy FFPE Kit as compared to the Promega ReliaPrep™ FFPE Total RNA Kit (2 μg vs 0.9 μg in average).
Results
The yield was further increased (65 %) by modification of the deparafinization procedure. Using random primers for cDNA synthesis combined with a pair of qPCR primers targeting a 100-bp sequence spanning two exons, we found that AID mRNA was consistently and reproducibly detected in both recently obtained and old FFPE samples. Applying our method to artificially degraded RNA extracted from B-cell line cells, we found that AID mRNA levels were not changed when the RNA integrity number (RIN) varied between 2.1 and 10. Applying this criterion to archived FFPE samples from patients with FL that had been stored for between 0.5 and 5 years allowed us to successfully quantify AID mRNA levels in 33/56 (59%) cases. To validate our findings, levels of AID mRNA were compared to levels of AID protein detected in all of the 33 samples by IHC; a significant positive correlation (P < 0.001) was observed.
Summary
In summary, we have shown that a sufficient quantity of mRNA for RT-qPCR can be extracted from only two sections of 4mm-thick FFPE tissue if an optimised protocol is applied. Our findings are relevant not only to the measurement of AID but to all situations where mRNA quantification is required and tissue availability is a limiting factor.
Session topic: Publication Only
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