Journal Article Summary
“Non-invasive lung cancer diagnosis by detection of GATA6 and NKX2-1 isoforms in exhaled breath condensate” by G. Barreto et al.
The RTube has successfully been used to isolate RNA, microRNA and DNA from Exhaled Breath Condensate (EBC). While the collection process of EBC is similar for each of the target isolates, the processes for isolation vary. This Journal Article Summary provides an example of nucleic acid isolation from EBC collected with the RTube based on a study conducted by Dr. Guillermo Barreto of the European Molecular Biology Organization (EMBO Medicine). Hyperlinks are present throughout the article to allow the reader to easily view the page for each product used during these experiments. This study utilized the RTube for the collection of EBC in order to isolate RNA from the EBC of lung cancer patients. This particular study was focused on the GATA6, and NKX2-1 genes in an effort to develop successful methods of detecting early-stage lung cancer.
EBC was collected using the RTube and following the guidelines for EBC by the ERS/ATS Task Force. All donors were asked to refrain from drinking any liquid (except water) for 2 hours before the collection of EBC. To avoid oral, or nasal contamination, the donors were asked to rinse their mouths with freshwater before collection, and to wear a nose clamp during collection. The donors were asked to use tidal breathing into the mouthpiece for 10 minutes. After the 10 minute period of breathing is over, the samples were immediately stored in 500 µl aliquots and cooled to -80oC. It is critical to freeze the samples as soon as possible after collection. EMBO medicine used a medical freezer to obtain this temperature. Due to the instability of RNA, without the aid of an RNase inhibitor, the samples will quickly deteriorate in the aliquots. To prevent the denaturing of RNA, EMBO medicine treated the microcentrifuge tubes with RNaseZap, and then autoclaved them twice prior to filling with condensate samples. It is very important to conduct the whole process under RNase free conditions. This includes barrier tip filters, and washing all equipment and gloves with RNaseZap. Gloves must be worn. If they are not, enzymes from the skin will denature the sample.
To isolate RNA from the autoclaved sample, 500 µl of EBC was used by EMBO medicine, and then treated with the RNeasy micro kit . The High Capacity Complimentary DNA (cDNA) Reverse Transcription kit was used to synthesize cDNA. 0.5–0.7 µg of EBC was used by EMBO Medicine in this step. As a negative control, a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was done without enzyme addition. Quantitative RT-PCR was done using SYBR® Green on the first step as well as Real-Time PCR Kit using the primers listed on page 13 in the appendix of this journal article. Briefly, 1× concentration of the SYBR Green master mix, 250 nM, each forward and reverse primer, and 3.5 µl (EBC) from an RT reaction diluted sixfold [0.16666667]were used for the gene-specific qPCR processes. Ratios of expression of the housekeeping genes (GAPDH and HPRT1), using two different primer pairs that were complementary to different segments of the matching mRNA were used to determine mRNA integrity and performance of the RT reaction.1
This study utilized two negative control groups. One was to add water instead of cDNA (complementary DNA) in the PCR, and cDNA from a control template that would be certain to give a negative result. In this situation, cDNA from control lung tissue was used. A total of three positive control groups were used. Serial dilutions (1000 to 1 copy) of cloned-PCR product containing plasmids were used as “calibrator” to form an accurate liner range of the system. The second positive control, referred to as the “analytical standard” was cDNA from human lung cancer cell lines H520 and A549; this scenario should unequivocally yield a positive result. The final positive result in the study conducted by EMBO Medicine was the use of cDNA from human LC biopsies that was used as the “biological standard” that unequivocally provides a positive result. Triplicates were done for every PCR test, and the PCR results were then normalized with respect to the gene TUBA1A.
Dr. Barreto and his team were able to successfully isolate DNA and RNA from cells gathered off of the lining of the lungs through EBC. Respiratory Research Inc. is constantly following up with our clients’ studies and results in order to provide guidance for future studies. This study was conducted by Dr. Barreto with intentions to solve broad health issues and to provide a better life for those who suffer from early stage lung cancer.
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