Learn More. Heymanslaan 10, Ghent, Belgium. The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Obtaining sufficient RNA yield and quality for comprehensive transcriptomic studies is cumbersome for clinical samples in which RNA from the pathogen is present in low numbers relative to the nucleic acids from the host, especially for pathogens, such as yeasts, with a solid cell wall.
Therefore, yeast cell lysis including cell wall disruption constitutes an essential first step to maximize RNA yield. Moreover, during the last years, different methods for RNA extraction from yeasts have been developed, ranging from classic hot phenol methods to commercially available specific kits.
We observed that, for C. Cell lysis efficiency was decreased to In our hands, the most efficient cell lysis and highest RNA yield from C. The online version of this article Candida albicans is the main infectious agent responsible for oral and vaginal candidiasis [ 1 ].
The fungus is usually a harmless inhabitant of the mucosal surfaces, but the loss of local host defense mechanisms together with the intrinsic virulence factors of Candida spp. Occasionally, the fungus can reach the bloodstream and the infection turns into a systemic disease, becoming fatal in immuno-compromised individuals [ 3 ]. The need for faster diagnostic tools in life-threatened patients, the high rate of recurrence in which patients suffer new episodes of candidiasis after anti-fungal therapy and the continued development of Next-Generation Sequencing techniques have boosted the study of host-pathogen interactions.
Several reports have focused on transcriptomic analysis for a better understanding of the genes involved in these host-pathogen interactions [ 4 , 5 ].
Although much has been elucidated about the human response, better understanding of the expression of fungal genes, which are in a very low proportion as compared to the human counterpart, is still lacking. There have been different attempts to resolve this question. The use of Candida -specific probes, that hybridize with all mRNAs of Candida , including the different splicing variants, to enrich fungal RNA is a promising method to improve our knowledge on this topic [ 6 ].
There are many reports comparing different methods for RNA extraction in different microorganisms such as viruses [ 7 , 8 ], bacteria [ 9 , 10 ], and also fungi [ 11 , 12 ]. The latter require additional cell wall disruption, and therefore more complex cell lysis approaches. Cell wall disruption methods are mainly focused on bead beating and enzymatic treatment and their efficiency has been shown to vary even with cell cycle [ 13 ].
Whereas most studies on Fungi deal with Saccharomyces cerevisiae , methods for maximal cell lysis efficiency, a crucial step prior to RNA extraction, and for RNA extraction, have not been reported in C. Cell lysis itself is not only important to increase yields of nucleic acids incl. In this study, we compared different cell lysis methods and evaluated cell lysis efficiency through microscopic visualization. In addition, we studied the effect of storage of cells in RNAlater on the efficiency of cell lysis and RNA extraction.
To evaluate which of different cell lysis methods and commercial RNA extraction kits were the most efficient to obtain high yield and high quality RNA from yeast cells, we prepared 1-ml aliquots, each containing 10 7 C. We compared lysis of thawed aliquots with Lyticase in Lyticase Lysis Buffer LLB without bead beating - as the control method proposed to yield very efficient lysis - vs bead beating in four different lysis buffers.
We also carried out bead beating in two different vortexes, a normal vortex in which tubes are oriented vertically and a hands-free vortex that allows bead beating in a horizontal position and thus increases the lysis area. Subsequently, we evaluated microscopically the percentage of cell lysis that could be obtained with the different treatments.
A general outline of the study is depicted in Fig. A schematic overview of the study set up for a cell lysis and b RNA extraction. Cell lysis is a crucial step during RNA extraction since the amount of lysed cells impacts directly on the amount of RNA obtained at the end of the process.
Here, we tested cell lysis efficiency by using four different lysis buffers, i. This adapter allows hands-free vortexing and also bead beating in a horizontal position, increasing the surface area for shearing and lysis. Percentage of cell lysis obtained after different treatments. Error bars represent the standard deviations of results from six biological replicates. We concluded that vortexing alone is not sufficient and that bead beating is needed to lyse yeast cells properly.
As we observed that bead beating in a horizontal position was an essential step for the most effective lysis of yeast cells see Bead beating vs vortexing without beads in RPLB and saline , we wondered about the importance of the horizontal orientation of the tubes for the lysis of Candida cells. After having shown that horizontal bead beating was more efficient than vertical bead beating, that bead beating was more efficient than vortexing alone and that RPLB was the most efficient lysis buffer, we next asked whether enzymatic treatment with lyticase would be more efficient than bead beating to lyse yeast cells.
RNAlater is an RNA protective agent that has been shown to perform equally well as snap-frozen methods to stabilize transcriptomic profiles [ 15 ].
CT values were plotted. Each dot represents the CT value for a technical triplicate. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing.
Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Buffer RLT. Catalog Number:. DO NOT add bleach or acidic solutions directly to the sample-preparation waste. Guanidine hydrochloride in the sample-preparation waste can form highly reactive compounds when combined with bleach. Reorder now! Reorder from your past orders in just one click.
Order by Catalog Number. Catalog Number. Pre-hybridization buffer contains BSA and will leave a large streak down the slide, and ruin the subsequent scans. Make sure that the solution covers the slides. Wash the slides twice by dipping up and down around 10 times each in MilliQ water in clean wash vessels.
Wash the slides once by dipping up and down around 10 times in RT isopropanol. Spin dry the slides at 1, rpm for 5 mins at RT. NOTE: Make sure the slides are used within one hour of spinning dry.
Atmospheric humidity will cause problems. Dip lifter slip in clean dH2O ii. Blow dry with clean air. You may use a Kimwipe to remove any other blotches you are unable to remove by the above method Hybridization i. After analyzing your labeled samples by absorbance, take 20 pmol of Cy3-labelled sample and 20 pmol of Cy5-labelled sample and combine the samples to be hybridized on one slide together.
Make up hybridization solution master mix for all the slides. Keep the sample on ice for at least 1 min. Remove it from ice, spin down quickly using table top microcentrifuge and keep samples covered from light at RT or warmer until ready to put on slide.
Place the pretreated cover slips on slides, one at a time xi. Pipet all of the sample onto the spotted side of the pretreated slide under the short side of the lifter slip. Seal Hybridization Chamber. Washing i. Immerse the pair of slides in 1 X SSC and slide the arrays past one another. Wash the slides in 1X SSC for 3 mins followed by wash in 0.
Keep some kimwipe on the bottom of the tube, place barcode facing the bottom of the tube and place in centrifuge with barcode facing out. Spin dry the slides at 1, rpm for 5 min in room temp in the 50 ml tube. Store the slides in dark before scanning and analysis. These factors will influence how much input RNA is used, whether one or two rounds of amplification should be done, and how long to incubate the IVT reaction. Alternatively 10— ng of poly A selected RNA can be used in the procedure.
Tailor both the amount of input RNA used and the amplification procedure to produce the amount of aRNA needed for your microarray hybridizations. When amplifying small RNA samples however, e. RNA samples should be free of contaminating proteins, DNA, and other cellular material as well as phenol, ethanol, and salts associated with RNA isolation procedures. Impurities can lower the efficiency of reverse transcription and subsequently reduce the level of amplification.
An effective measure of RNA purity is the ratio of absorbance readings at and nm. The ratio of A to A values should fall in the range of 1. There are currently no quantitative methods for measuring what percentage of mRNAs in a sample are full-length, however several procedures do exist for establishing the relative integrity of a sample.
Denaturing agarose gel electrophoresis and nucleic acid staining can also be used to separate and visualize the major rRNA species. The primary drawback to gel electrophoresis is that microgram amounts of RNA must be sacrificed. Reaction Incubation times should be precise and consistent The incubation times in the protocol were optimized in conjunction with the kit reagents to ensure the maximum yield of nucleic acid product in each step—adhere to them closely. Refer to the graph in Figure 3 on page 7 to help determine what incubation time to use.
Keep this IVT incubation time uniform if aRNA yield from different samples will be compared, or if you want to have equal amplification of different samples. Although differences in IVT incubation time among samples has had very little, if any, effect on array results in our hands, we still recommend using a uniform IVT incubation time for the most reproducible amplification and array analysis.
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