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Fulltext Alternative RNA Extraction Method in Vibrio vulnificus Infected BrownMarbled Grouper, Epinephelus fuscoguttatus

Fahmeeda Mohamad Jazamuddin, Wan Mohd Aizat, Hoe-Han Goh, Chen-Fei Low, Syarul Nataqain Baharum

Trop Life Sci Res, 2019;30(2):2012-209.
MyJurnal

Vibriosis is a prevalent aquatic disease caused by Vibrio species and has led to massive loss of brown-marbled grouper, Epinephelus fuscoguttatus. The complexity of molecular mechanisms associated with immune defence can be studied through transcriptomics analysis. High quality and quantity of total RNAs are crucial for the veracity of RNA sequencing and gene expression analysis. A low quality RNA will compromise downstream analysis, resulting in loss of time and revenue to re-acquire the data again. Thus, a reliable and an efficient RNA isolation method is the first and most important step to obtain high quality RNA for gene expression studies. There are many aspects need to be considered when deciding an extraction method, such as the cost-effectiveness of the protocol, the duration of chemical exposure, the duration required for a complete extraction and the number of sample-transferring. A good RNA extraction protocol must be able to produce high yield and purity of RNA free from enzyme inhibitors, such as nucleases (RNase), phenols, alcohols or other chemicals carryover, apart from protein and genomic DNA contamination, to maintain isolated RNA integrity in storage condition. In this study, TransZolTM Up produced clean and pure RNA samples from control gills only but not from the infected gill and whole-body tissues. Modified conventional CTAB (conventional hexadecyltrimethylammonium bromide) method was then used as an alternative method to isolate RNA from gill and whole-body tissues of Vibrio-infected E. fuscoguttatus. Modified CTAB method produced intact RNA on gel electrophoresis with higher RIN number (>6.5) for infected gill and whole-body tissues, suggesting that this method could also be used to isolate high quality RNA from fish samples. Therefore, this method is potentially suitable to be used to extract RNA from other fish species especially those that have been infected.
Optimization method for proteomic analysis of the larva and adult tissues of plutella xylostella (l.) (lepidoptera: plutellidae)

Maizom Hassan, NorazilaYusoff, Wan Mohd Aizat, Idris Abd Ghani, Nurul Wahida Othman

Sains Malaysiana, 2018;47:2975-2983.

Plutella xylostella (L.) (Lepidoptera: Plutellidae), the major insect pest of cruciferous crops worldwide shows significant
resistance to almost all classes of insecticides. In order to effectively prevent and manage the insecticidal resistance,
it is crucial to understand the physiological adaptation of insects against insecticides. Identification of insect protein
that interacting with insecticides and characterization of their modification in resistant strains can be done by using
differential proteomics approach. This study focuses on optimizing a sensitive and rapid method for the extraction of
high quality protein of both larva and adult tissues of P. xylostella to be used in two-dimensional gel electrophoresis.
Five extraction methods were evaluated for protein concentration, yields and resolving patterns of one-dimensional
and two-dimensional electrophoresis. The results showed that trichloroacetic acid/acetone extraction methods with
two different concentrations of 2-mercaptoethanol produced the highest protein concentration and yield for both adult
and larva tissues, respectively. Meanwhile, trichloroacetic acid/acetone with dithiothreitol extraction method gave
better separation of spots and intensity for both larva and adult tissues compared to other methods tested. As such, we
concluded that trichloroacetic acid/acetone with dithiothreitol successfully yielded high total protein concentration and
good separation of two-dimensional electrophoresis gel spots in both adult and larva P. xylostella.
Proteomic analysis of stored core oil palm trunk (COPT) sap identifying proteins Related to stress, disease resistance and differential gene/protein expression

Marhaini Mostapha, Noorhasmiera Abu Jahar, Sarani Zakaria, Sharifah Nabihah Syed Jaafar, Kamalrul Azlan Azizan, Wan Mohd Aizat

Sains Malaysiana, 2018;47:1259-1268.

Oil palm is the major crop grown and cultivated in various Asian countries such as Malaysia, Indonesia and Thailand.
The core of oil palm trunk (COPT) consists of high sugar content, hence suitable for synthesis of fine chemicals and
biofuels. Increase of sugar content was reported previously during prolonged COPT storage. However, until now, there
has been no report on protein profiles during storage. Therefore, in this study, protein expression of the COPT during the
storage period of one to six weeks was investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) coupled with optical density quantification and multivariate analyses for measuring differentially expressed
proteins. Accordingly, protein bands were subjected to tryptic digestion followed by tandem mass spectrometry (nanoLCMS/MS)
protein identification. The results from SDS-PAGE showed consistent protein bands appearing across the biological
replicates ranging from 10.455 to 202.92 kDa molecular weight (MW) regions. The findings from the principal component
analysis (PCA) plot illustrated the separation pattern of the proteins at weeks 4 and 5 of storage, which was influenced
mainly by the molecular weights of 14.283, 25.543, 29.757, 30.549, 31.511, 34.585 and 84.395 kDa, respectively. The
majority of these proteins are identified as those involved in stress- and defense-related, disease resistance, as well
as gene/protein expression processes. Indeed, these proteins were mostly upregulated during the later storage period
suggesting that long-term storage may influence the molecular regulation of COPT sap.
RNA extractions of mangosteen (garcinia mangostana l.) pericarps for sequencing

Azhani Abdul-Rahman, Najah Izzati Suleman, Wan Adibah Zakaria, Goh HH, Normah Mohd Noor, Wan Mohd Aizat

Sains Malaysiana, 2017;46:1231-1240.

This study employed several RNA extraction methods for mangosteen pericarps prior to RNA sequencing. The sequencing platform heavily relies on a high quality RNA yield. However, pericarp tissues contain a lot of phenolic compounds that results in low RNA quality. Hence, we studied several RNA extraction methods to obtain the most suitable method for the best RNA quality from the pericarps of mangosteen. Five different methods including Lopez and Gomez, modified hexadecyltrimethyl ammonium bromide (CTAB) method, several commercial kits from TranszolUP, Favorgen and Qiagen RNeasy were compared. By optimising the CTAB method, it was found to be the best method to obtain pure RNA (high A260/A280 ratio) with the highest yields (up to approximately 600-800 ng/μL concentration). The QC control of these samples using bioanalyzer validated their suitability for the downstream RNA sequencing. This report details the method for extracting high quality and high yield RNA samples from fruit that are rich in polyphenolic compounds such as mangosteen.
Fulltext Effect of Age on the Protein Profile of Healthy Malay Adults and its Association with Cognitive Function Competency

Abu Bakar ZH, Damanhuri HA, Makpol S, Wan Kamaruddin WMA, Abdul Sani NF, Amir Hamzah AIZ, et al.

J Alzheimers Dis, 2019;70(s1):S43-S62.
PMID: 30594926 DOI: 10.3233/JAD-180511

BACKGROUND: Many studies on biochemical and psychological variables have aimed to elucidate the association between aging and cognitive function. Demographic differences and protein expression have been reported to play a role in determining the cognitive capability of a population.
OBJECTIVE: This study aimed to determine the effect of age on the protein profile of Malay individuals and its association with cognitive competency.
METHODS: A total of 160 individuals were recruited and grouped accordingly. Cognitive competency of each subject was assessed with several neuropsychological tests. Plasma samples were collected and analyzed with Q Exactive HF Orbitrap. Proteins were identified and quantitated with MaxQuant and further analyzed with Perseus to determine differentially expressed proteins. PANTHER, Reactome, and STRING were applied for bioinformatics output.
RESULTS: Our data showed that the Malay individuals are vulnerable to the deterioration of cognitive function with aging, and most of the proteins were differentially expressed in concordance. Several physiological components and pathways were shown to be involved, giving a hint of a promising interpretation on the induction of aging toward the state of the Malays' cognitive function. Nevertheless, some proteins have shown a considerable interaction with the generated protein network, which provides a direction of focus for further investigation.
CONCLUSION: This study demonstrated notable changes in the expression of several proteins as age increased. These changes provide a promising platform for understanding the biochemical factors affecting cognitive function in the Malay population. The exhibited network of protein-protein interaction suggests the possibility of implementing regulatory intervention in ameliorating Malay cognitive function.