Synthesis of good quality double-stranded cDNA from the bark tissue of robusta coffee (Coffea canephora) plants

Quality RNA in large quantity is often required in the analysis of gene expression. RNA extraction from samples collected from woody plants is generally complex and becomes the main limitation to study gene expression particularly in perennial crops like coffee. Standard RNA extraction protocols are time consuming laborious and cannot be adapted for high throughput functional analysis. A simple and effective protocol for extraction of high quality total RNA from bark tissue of woody stem was achieved using the RNeasy plant mini kit (Qiagen, USA). The extracted RNA was successfully converted into double-stranded cDNA using the SMATer cDNA synthesis kit (Clontech, USA) which is based on the Switching Mechanism At 5’ End of RNA Transcript (SMART) technology. The integrity  of the total RNA used for synthesizing double stranded cDNA was assessed by amplifying a 1282 bp product targeting the glyceraldehyde 3-phospho dehydrogenase (GAPDH) gene by PCR. As expected, the PCR product contained the full coding sequence plus 69 and 196 bp of 5’ and 3’ UTRs respectively. The double-stranded cDNA was used successfully for creating  a SSH cDNA library (results not reported here). The cDNA could also be useful for a number of other applications like cDNA library construction, EST analysis, RACE and Next Generation Sequencing (NGS)

A simple method of DNA extraction from coffee seeds suitable for PCR analysis

High quality genomic DNA was successfully extracted from coffee seeds using a simple protocol devoid of liquid nitrogen or freeze-drying and proteinase K. The isolated DNA was quantified using spectrophotometer and using agarose gel electrophoresis. The DNA was free from polysaccharides, polyphenols, RNA and other contaminants. The quantity of DNA ranged from 180 to 630 g/g of seed powder. Quality of DNA was confirmed by digestion using EcoRI, HindIII and PstI restrictionendonucleases and complete digestion was observed. PCR with random decamer primers and consensus primers of mitochondria and chloroplast DNA and PCR-RFLP revealed the suitability of the DNA for PCR based marker techniques including diagnostics

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

Effect of genotype on chromosome variation in tissue-culture of inbred and outbred rye

To examine the effect of genotype on the nature of the cytological variation induced in culture, plants were regenerated from cultured immature embryos of six genotypes (lines A-F) of rye (Secale cereale L.). The variation observed differed among the six lines and was generally consistent with the donors. Regenerants of line A were consistent with the donor, cv. Ailes, in that chromosome translocations appeared at high frequencies. No structural or numerical variation was observed in the B chromosomes of line B. Two of the four inbred lines studied were very stable in culture. Inbred line D was also stable except for a translocation in the regenerants. In inbred line F, variation was observed in the donors and the regenerants. An examination of chiasma frequency and distribution indicated that the regenerated plants generally had higher chiasma frequencies and more distally localized chiasmata than the donors. Line F was exceptional in that the regenerants had more interstitial chiasmata. The elevation in chiasma frequency was not as great, or statistically as significant, as the change in distribution. These results provide further evidence that the nature of cytological variation observed in regenerated plants is genotype-dependent and that, in addition to numerical and structural chromosome variation, shifts in chiasma frequency and distribution, and therefore in genetic recombination, can occur as a result of somaclonal variation