ABSTRACT
The growing interest in biomedical studies has brought RNA from biofluids including plasma, as
promising candidates for genetics profiling. The precision and reliability of an analysis in
downstream application such as NanoString nCounter® MAX Analysis System (NanoString
Technologies, Seattle, WA) ) depend on the RNA quality, purity and level. In this project,
NanoString nCounter® miRNA panel was chosen due to rapid identification and ability to profile
approximately 800 miRNAs per run which requires total RNAs from plasma with a minimum
concentration of 33.3 ng/µL with 260/280 and 260/230 ratios of ≥1.8 for optimal results. Unlike
tissues and cells, circulating RNAs in plasma are cell-free and are present in small sizes. However,
the abundance of proteins and inhibitors in the plasma as possible contaminants could diminish
the effectiveness of molecular isolation techniques and pose challenges in RNA isolation and
quantification. This could skew data collection and elucidation. Therefore, the main objective is
to determine the optimized plasma RNA isolation protocol to overcome problems in RNA quality
and purity with regards NanoString nCounter® MAX Analysis System requirement. Several
optimization steps were performed, including the addition of one chloroform extraction step with
extra washing steps instead of conducting only once following the actual protocol. After
conducting these steps, the average 260/280 ratio falls between 1.7 to 1.8, slightly increased
compared to the results before optimization which was around 1.4 to 1.6 since these steps of
optimization help to remove excess impurities including phenol and salt. Furthermore, increasing
the incubation time in certain steps, for instance, after sample homogenization with Qiazol, during
95% ethanol precipitation and after RNase-free water addition have boosted the RNA recovery
allowing RNA concentration of 15 ng/µL and above to be obtained. Hence, the optimized plasma
RNA isolation protocol was determined since several issues related to plasma RNA concentration
and purity were significantly improved by performing the additional steps in the protocol.
MGMT (O6
-Methylguanine-DNA Methyltransferase) suppresses tumor development by removing alkyl adduct, while
SPOCK2 (SPARC/Osteonectin CWCV and Kazal-like domains proteoglycan) abolishes the inhibition of membrane-type
matrix metalloproteinases (MT-MMP) which leads to angiogenesis. Hence, MGMT methylation may initiate malignant cells
transformation. In contrast, SPOCK2 methylation is hypothesized not to be a common event in diffuse large B-cell lymphoma
(DLBCL). In this study, we examined the methylation status of MGMT and SPOCK2 in DLBCL as in Malaysia the information
is extremely lacking. A total of 88 formalin-fixed paraffin-embedded tissue of patients diagnosed with DLBCL from the
year 2006 to 2013 were retrieved from Hospital Universiti Sains Malaysia, Kelantan and Hospital Tengku Ampuan Afzan,
Pahang. Methylation-specific polymerase chain reaction (MSP) was used to examine the methylation status of both genes.
Interestingly, methylation of MGMT was detected in all the 88 DLBCL samples, whereas SPOCK2 was found to be methylated
in 83 of 88 (94.3%) DLBCL cases. Our study showed a remarkably high percentage of promoter methylation of both
MGMT and SPOCK2 genes. Our finding also negates initial expectation that SPOCK2 methylation would be an uncommon
event in the majority of DLBCL cases. This study has shown a very high percentage of promoter methylation of MGMT and
SPOCK2 in the DLBCL cases studied by MSP, using archival lymphoma tissues. Nonetheless, additional research is needed
to quantitatively evaluate MGMT and SPOCK2 methylation, and to analyse gene expression and/or protein expression in
order to further understand the role of MGMT and SPOCK2 methylation in the pathogenesis of DLBCL.