Figure 1: Yeasts with preserved cell wall, prior to the action of zymolase. (Unstained sample, 40X o.m.)
Figure 2: Yeast with altered cell wall, 2 hours after the action of zymolase. (Sample stained with toluidine blue, 40X o.m.)
Figure 3: The ITS1 and ITS4 primers allow raising a region of the fungal ribosomal RNA of 517bp
Figure 4: The Zymo research system using tri-reagent as a lysis buffer generated the highest values of RNA concentration, with a significant difference over the qiagen system. Difference of means: 0.78 (95%CI: 0.37-1.20); p-valor: 0.00054
Figure 5: Average and standard error for absorbance ratios 260/280 and 260/230 of both commercial systems
Note: L: Ladder DNA; Street 1 to 8: RNA extracted and purified with Qiagen system; Street 9 to 16: RNA extracted and purified with Zymo-research system; Streets 5,6,12,13,14,15,16 with degradation.
Figure 6: To evaluate the integrity of the extracted RNA, it was run on 2% agarose gel, 8 samples extracted with Qiagen system, and 8 samples extracted with Zymo-research system, chosen at random.In each street 10uL of RNA was seeded with a concentration equivalent to 5ug of RNA. Note the absence in all lanes (1-16) of genomic DNA
Note: Sample 10 throws a band of smaller size to the others. Probably it is a different genotype of the same species, since the strain is confirmed by sequencing.that it is Candida parapsilosis sensu stricto. Figure 7: Electrophoretic run of PCR products using primers ITS1-ITS4. L=Ladder DNA 100pb; Street 1=Negative control; Street 2=Positive control, with genomic DNA (10uL/100ng) of reference strain (ATCC 22019); Street 3=Detection limit control with sample cDNA (1uL/1ug); Streets of 3 to 10=cDNA of samples processed with Qiagen ; Streets of 11 to 18=cDNA of samples processed with Zymo-Research
Variable
QIAGEN SYSTEM
ZYMO-RESEARCH SYSTEM

Mean

S.E

95%CI(Mean)

Mean

S.E

95%CI(Mean)

RNA (ug/uL)

1,3

0,15

0,99-1,61

2,08

0,14

1,78-2,38

Abs 260/280

2,04

0,03

1,99-2,1

1,83

0,06

1,71-1,95

Abs 260/230

1,53

0,15

1,23-1,84

1,99

0,07

1,83-2,14

Table 1: Descriptive and inferential statistics for the yield and purity variables with each commercial system tested

Variable

U

p(2tails)

Abs(260/280)

206

0,0003

Abs(260/230)

393,5

0,0117

Table 2: Model coefficient estimated by multiplies linear regression

Table 3: Screenshot of the result thrown by PrimerBLAST about the features, sequence and specificity of the primers used for RT-PCR
Table 4: Screenshot of the results obtained by the BLASTn algorithm on the identity of the PCR product obtained with the ITS1-ITS4 primers
 
Lysis system
Mechanism of action
Advantages for the target and operator
 Disadvantages for the target and operator

Zymolase
(Querol et al. 1992) [36]
(Klassen et al. 2008) [43]
(Suzuki et al. 2013) [45]
Enzymatic: hydrolyzes glucose polymers linked by ß-1,3-bonds, producing laminaripentaose.
High efficiency.. No toxicity, and avoid the use of phenol chloroform. Does not affect the integrity of RNA.
 Consumption of time, high cost. According to Suzuki et al. enzymatic treatment can generate changes in gene expression. However this has not been validated.

Beta-Glucoronidase
(Cadavid E, et al. 2009) [46]
Enzymatic: catalyzes the reaction Beta-D-glucuronoside+ H2O ↔ D-glucuronate+ Alcohol
Fast obtaining of DNA, in good concentration and of high quality. Decrease in time and costs.
 There are no experiences with RNA.

Betamercaptoethanol
(Nelson et al. 2005) [47]
Chemical: reducing agent, reduces disulfide bridges.
Irreversibly denatures ribonucleases. Protects the RNA
 It`s toxic for to the operator. Requires combining with another method

SDS: sodium duodecyl sulfate
(Rojas et al. 2011) [48]
(Rodrigues et al. 2017) [41]
Chemical: ionic detergent, denatures proteins.
It does not affect the integrity of the RNA. No toxicity and low cost.
 Contaminates the RNA with DNA. Requires DNase purification. Inhibits PCR at minimal concentrations. Requires combining with another method.

CTAB: hexadecylmethylammonium bromide
(Rodrigues et al. 2017) [41]
(Sandoval et al. 2017) [25]
Chemical: detergent.
Reduce contamination with carbohydrates. No toxicity. Low cost.
 It does not protect the RNA from degradation. Time consuming. It requires combining with another method.

Trizol/Tri-reagent
(Phenol, chloroform, more guanidine isothiocyanate) (Chomczynski 1993) [49]
(Sandoval et al. 2017) [25]
Chemical: Denatures and removes proteins.
It is an RNA stabilizer, Inhibits RNases. Good quality and integrity of RNA extracts have been reported in Sacharomyces ceriviciae model
 It is toxic, requires cabin management and protection barriers. It can generate contamination with carbohydrates. It decreases the performance of the PCR at concentrations of 0.2% and 0.5% completely inhibits it. High cost and time consuming. Requires combining with another method.

Glass beads
(Hoffman y Winston, 1987) [50]
Mechanical: breaks the cell wall by hitting.
No toxicity for the operator. Low cost.
 It can compromise the integrity of the RNA. Requires technique and combination with another method.

Sonication
(Muller et al. 1998) [40]
Mechanical: ultrasonic waves to stir particles
Suitable for all cell types and easily applicable in small and large scale. Save time.
  

Table 5: Mechanism of action, advantages and disadvantages of the lysis systems most commonly used in fungi

Qiagen System
(RNeasy Mini Kit)

Zymo-research system
(Direct-zol™RNA MiniPrep)
Inoculum size
1-2x107
1-2x107sup>

Protein denaturation and  inhibition of RNasas
Isotiocianato of guanidina
Isotiocianato of guanidine +  Phenol

Elimination of  carbohydrates
It is unknown
Phenol
Precipitation of RNA
Column
Column
Toxicity
Low
High
Extracted material
Total RNA
Total RNA
Prize in the Argentina for 50 columns
900 USD
500 USD

Table 6: Differences between Qiagen and Zymo-research system for RNA extraction and purification Font: Hernández A et al, (2013) [23]