Chromosomal Microarray Analysis (CMA) & Fluorescence in situ Hybridization (FISH) to determine PD phase

Due to the relatively high frequency of copy number variation (CNV) within PRKN, chromosomal microarray (CMA) is a robust approach for the detection of clinically‐relevant genetic variants resulting in Early Onset Parkinson´s Disease (EOPD). However, CMA seems unable to determine whether CNVs occur on the same allele (cis) or on opposite alleles (trans) necessitating follow up studies. For this reason, Follow‐up fluorescence in situ hybridization (FISH) studies of the proband and parents demonstrate the types of deletions, providing a genetic etiology for the clinical diagnosis of EOPD (1).

Reference

1. Williams E, Barrett M, Dhamija R, Toran L, Chambers C, Mahadevan M et al. Phase determination using chromosomal microarray and fluorescence in situ hybridization in a patient with early onset Parkinson disease and two deletions in PRKN. Molecular Genetics & Genomic Medicine. 2018;6(3):457-462.

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Quantitative analysis of nasal transcripts using PCR for the find of potential biomarkers in PD

Figure 1. PCR and agarose gel showing the GAPDH amplicon

Figure 2. Table with the primers used during PCR

The aim of this study, was to investigate whether the transcripts from nasal fluid cell pellets obtained from subjects with PD were quantifiable and therefore could serve as potential biomarkers for the diagnosis of the PD status. The steps involved were as follows: 

First created a standardized protocol for mRNA purification from nasal lavage cells and validated whether PCR assessment was applicable. Subsequently, cell pellets from each subject were obtained from the centrifugation of the nasal lavage fluid collected from a single nasal irrigation. To minimize RNA loss, considering the small quantity of RNA from each nasal lavage, a carrier yeast tRNA was included to maintain the consistency in the total RNA purification steps. Successively, cDNA was synthesized by reverse transcription employing the total RNA as templates and subjected to PCR using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a target for validation. Finally, The GAPDH amplicon by PCR was visible under UV following the separation in agarose gel, indicating successful cDNA synthesis from the total RNA prepared from the two nasal fluid cell pellets (Figure 1) (1).

Summary

Total RNA was extracted from the nasal lavage cell pellet using the QIAzol Lysis Reagent (cat# 79306, QIAGEN) with the addition of a carrier yeast tRNA (10 µg) followed by DNase I treatment to eliminate any trace of DNA contamination. cDNA was synthesized from purified nasal total RNA employing the First-strand cDNA synthesis kit (iScript cDNA synthesis kit, Biorad). Ct values of each gene were obtained from the SYBR green-based real-time PCR reaction by using the QuantStudio 6 flex Real-Time PCR System (Applied Biosystems). Relative mRNA expression levels of target genes were calculated through the ΔΔCt method using GAPDH as the internal loading control. The SYBR green PCR master mix (Cat# 4309155, Applied Biosystems) was used according to the manufacturer’s instructions. The primers utilized for real-time PCR amplification are summarized in figure 2 (1).

Reference

1. Kim H, Kang S, Jo Y, Kim M, Lee Y, Cho S et al. Quantitative analysis of nasal transcripts reveals potential biomarkers for Parkinson’s disease. Scientific Reports. 2019;9(1).

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