Introduction

The use of molecular techniques for studies related to the expression of differential genes requires the synthesis of complementary DNA of good quality (^{Aslam et al., 2017}). In this way, the availability of a good quality RNA is the first requirement for the understanding of research in plants. Different methods have been described for the isolation of RNA using various detergents such as cetyltrimethylammonium bromide (CTAB) because it is economical, SDS, phenol, chloroform, lithium chloride and different centrifugation gradients (^{Bermúdez-Guzmán et al., 2016}; ^{Silva et al., 2016}). For its part, the preservation of the integrity of RNA during extraction is paramount.

The precise quantification of mRNA used for the gene expression profile depends on the integrity of the extracted RNA. Therefore, the use of poor quality RNA during the quantification of mRNA levels may compromise the accuracy of gene expression results (^{Bleitler et al., 2016}).

The extraction of tissue RNA from mesquite plants (Prosopis spp.), is difficult because it is a plant rich in secondary metabolites, polysaccharides and polyphenols (^{Ghangal et al., 2009}; ^{Chang et al., 2016}). The presence of compounds can affect the quality and quantity of the isolated nucleic acids (^{Abdel-Latif and Osman, 2017}). In turn, it is hindered due to differences in the content of metabolites present in different tissues (roots, stems and leaves) and stages of plant development (^{Aslam et al., 2016}). In addition, there are phenolic compounds that can be easily oxidized, which enables the formation of quinones due to their similar chemical properties, affecting the quality of the sample and the yield of RNA (^{Ghangal et al., 2009}; ^{da Luz et al., 2016}).

The advances in the cultivation of the passion fruit were obtained by genetic improvement. Then, the best method for extracting RNA in P. edulis should be determined so that analyzes such as the construction of complementary DNA library (cDNA), subtractive suppressive hybridization (SSH), RT-PCR, amplified fragment length polymorphism with cDNA (AFLP cDNA) and Northern blot are not limited by low quality samples

Prosopis juliflora (SW), commonly called ‘sweet mesquite’, is a tree belonging to the family Fabaceae and is a plant tolerant to drought, salinity, and heavy metals. It is widely distributed in arid and semi-arid regions. In Mexico, it is the most important species because of its size and uses, it occupies almost all Mexican territory, it grows in extreme climates with temperatures above 48 °C and in a variety of soils (^{Rodríguez-Sauceda et al., 2014}).

Materials and methods

Results and discussion

Currently there are published a large number of methods for the isolation of RNA from plants, in which some of the difficulties that exist during the extraction procedure have been mentioned. Generally, high-quality RNA from plant tissues can be obtained once the activities of the RNases are inhibited (^{Wang et al., 2008}). In the present study, we compared the results of three different protocols with the results of other protocols that were designed to extract RNA from plant tissues with high content of polysaccharides and polyphenols.

Obtaining high quality RNA is the basis of many investigations of plant molecular biology; RT-PCR, cDNA synthesis and subsequent genetic analysis, require RNA with high purity and integrity (^{Liu et al., 2018}). In Figure 1, the representation of the characteristic bands of the 28S and 18S subunits of the ribosomal RNA can be observed, thereby confirming the purification of the RNA. In turn, the quality of the total RNA was detected by the polymerase chain reaction. On the other hand, in this study, the use of polyvinylpyrrolidone (PVP) in the extraction buffer resulted in an improvement in the yield and quality of extracted RNA compared with the application of other methods for P. juliflora tissues (SW). ^{Wang et al. (2008)}, mention that PVP efficiently absorbs polyphenols and removes secondary metabolites of nucleic acids in plants, as well as most macromolecular substances.

Figure 1 The 1% agarose gel electrophoresis of the total RNA extracted from the tissues (leaf, stem, root and embryo) of mesquite P. juliflora (SW) of the three protocols evaluated. The two distinct bands, 18S and 28S of ribosomal RNA show that the RNA is intact (A, C) and (B) only samples of total genomic DNA were obtained. 

The phenolic compounds present in the tissue samples are rapidly oxidized to form quinones. These quinones can easily bind with nucleic acids and act as a barrier to the isolation of good quality RNA (^{George, 2018}).

The successful isolation and obtaining of an intact total RNA from tissues rich in polysaccharides and polyphenols is important to prevent the binding of contaminants to nucleic acids (^{Malnoy et al., 2001}). In turn, it has been reported that DNA contamination may not be completely avoided, however, the digestion of RNA with DNases free of RNases is essential; that is, digestion with DNases is a common step in the analysis of RNA protocols (^{Ghawana et al., 2011}).

The Trizol-based method has been widely used for the isolation of RNA from various tissues and species. First, we tried to use the Trizol reagent to isolate the RNA from the tissues of P. juliflora. However, the Trizol buffer could not efficiently remove the high content of metabolites causing the aqueous phase to turn red after purification with chloroform. However, protocol 2 was found to be favorable for the extraction of DNA from mesquite plant tissues.

Studies have shown that there are different factors that affect the amplification profiles obtained by molecular techniques based on PCR, some of the main factors that affect these amplification profiles are the quality, purity and quantity of the RNA extracted, which mainly has to do with the co-isolation of polysaccharides, phenols and secondary metabolites during RNA extraction (^{Solano-Florez et al., 2009}). In this study, the quality of total RNA was detected by the polymerase chain reaction. The RNA samples were reverse transcribed into cDNA and the result was amplified using PCR (^{Liu et al., 2018}). In this way, it is demonstrated that the total RNA extracted from P. juliflora through protocol 1 and 3 meet the requirements for future molecular research.

On the other hand, agarose gel electrophoresis shows that the extraction protocol implemented produced high quality RNA shown by the defined cytosolic and plasmid ribosomal RNA bands (^{Box et al., 2011}). The electrophoresis observed in Figure 2 indicates that the pair of primers used to amplify the actin gene fragment exhibits high specificity. The amplification curves showed that the mRNA sequences were virtually intact in the RNA samples and that the actin gene was present in all tissues in moderate abundance; in this way, it is demonstrated that the total RNA produced is of good quality for the RT-PCR analysis of constitutive genes such as actin with an approximate size of 300 bp (^{Kam-Lock et al., 2007}).

Figure 2 Electrophoresis in 1% agarose gel of the RT-PCR products of the actin gene with extracts of all tissues evaluated with the three extraction protocols and with plant-specific primers (expected size 300pb). For the three gels MP corresponds to the molecular weight marker GeneRuler 1Kb Plus DNA Ladder (ferment). 

The large number of original and modified methods is well known, which mostly report modest yields and partially intact RNA, which cannot be generalized or applied to different species, especially for RNA extraction, where the content of this nucleic acid is relative to the moment and state of the cells to be evaluated. In this study, the absorbance radii of all the analyzed samples were greater than 1.8, indicating a weak contamination produced by polysaccharides and polyphenols (Table 1), these results are similar to those reported by ^{Malnoy et al. (2001)} in species of the Rosaceae family.

Reliable and proven methodologies, sometimes they cannot be implemented in all laboratories, due to the cost of certain reagents or the difficulty of their acquisition in the laboratory. Although the results of the evaluated protocols differ, samples were obtained that fulfilled the characteristics of yield and quality, which can be used in the application of more advanced molecular techniques.

Conclusions

In conclusion, a rapid and efficient method for extracting RNA from tissues of plants with a high content of polysaccharides and polyphenols has been developed, as is the case of P. juliflora (SW). The RNA obtained by this method can be used for the detection of specific RNAs. Also, this method can also be applicable for the extraction of other tissues in different plants with characteristics similar to P. juliflora (SW).

In turn, with this document, we offer the possibility of an efficient, fast, safe and economic protocol for the simultaneous extraction of DNA and RNA, thus allowing the extraction of high quality genetic material, as well as the simultaneous extraction of nucleic acids, can be an advantage in the case of material of limited origin or to minimize the variation of the sample when the DNA has to be compared with the RNA of the same source or biological condition.