LOSS OF AN ABUNDANT QUANTITY OF RIBONUCLEIC ACID DURING MINI COLUMN ISOLATION METHOD

Aim . The Isolation of nucleic acid is an important step for conducting different molecular assays in many laboratories around the world. It is also a common practice that user is isolating the ribonucleic acid (RNA) from the samples with mini column once and throwing away the supernatant. This makes isolated RNA as limiting factor in many studies as this issue has not been addressed in literature. Therefore, we decided to conduct whether it is a loss of ribonucleic acid during the mini column isolation method. Method. In this research, we have made analysis whether there is a loss of RNA during the isolation process. The mini column isolations were done with different samples of human tissues from placenta and umbilical cords. These were assessed with spectrometric instrument and real time PCR machine. Results . It was found that there is loss of abundant quantity of RNA during the subsequent isolations. The amount measured with UV spectrometer indicates that some times 2 nd and 3 rd isolation have more RNA than the first isolation. Realtime PCR for house keeping gene beta actin shows that presence of RNA can be seen up to 6 isolation cycles from supernatants. Conclusion . There is loss of RNA in subsequent isolations with mini column method, therefore it is possible to isolate more RNA from the next supernatant isolations. User should do the multiple isolations to get higher yield of RNA.

Isolation of nucleic acid is an essential step for conducting the different nucleic acid analysis methods like PCR and hybridization for various viral, bacterial, biomarkers and genetic targets [1][2][3][4]. Not only this, the nucleic acid can be used as therapeutic as well as vaccine options in many clinical settings, therefore there is an urgent need to isolate the sufficient amount of nucleic acid in highly pure form [5][6][7][8]. Ribonucleic acid (RNA) can be isolated from different sources e.g., blood, tissue, buccal swabs, nasal samples, plasma, serum, urine and cell cultures. There are different methods available to isolate the RNA, which are magnetic beads, solutionsbased and mini column methods for examples. The mini column isolation method is widely used in the laboratories througaround the world. Usually most of the laboratories isolate the RNA from the sample once and the supernatant of this method is discarded.
Many times, there is limited volume of isolated RNA e.g., 50 or 100 μl available, which is not sufficient to conduct the whole studies, where user has to conduct a set of experiments on the same isolated RNA (homogenous material). In case of development of vaccines, one needs sufficient quantity of RNA, which may not be available from a single isolation source [4,9,10].
In this study, the experiments are conducted to find whether RNA is still in supernatant and this surplus RNA can be isolated to conduct the further analysis.
A protocol of local ethics committee approval of Genekam Biotechnology AG (Number: GEN01-23) about conducting the experiments of this research work is available. In this experiment, 4 samples from different tissues like umbilical cord, placenta tissue, placenta membrane etc. are used to isolate the RNA with mini column RNA isolation kit (Genekam, Germany). 100 μl of the samples were added to lysis buffer with proteinase K. These samples were kept at 56 C for 10 min. After that guanidine solution was added, while keeping at 70 C for 5 min. 400 ul of molecular ethanol (Applichem, Germany) was added to each well.
Mini column step. These mixtures were added to mini columns, which were centrifuged at the speed of 11000 rpm for 1 minutes. The supernatants from each sample were collected in separate tubes. Mini columns were washed with 500 μl B and C tubes each while centrifuging at the speed of 11 000 rpm for one minute. At the end mini columns were centrifuged in order to make them dry at the speed of 13 000 rpm for 1 minute. At last, 70 C warm elution buffer was added to each mini column, while keeping this for 2 minutes at room temperature. In order to get the elution buffer containing RNA, the mini columns were centrifuged. The tubes were labelled as first isolation and stored at 4 C for further analysis.
Collected supernatants were processed as mentioned in mini column step till there was use of elution buffer at the end to elute RNA. During this step, the supernatants were collected and processed again as mentioned above. There were two groups. In one group, there were 2 isolations (one original isolation and 1 isolation from supernatants, these are labelled as Isolation 1 and 2) and in 2 nd group, there are 7 isolations (one original isolation and 6 subsequent isolations from supernatants, which were labelled as Isolation 1 to 7). In both groups, isolation 1 is the original and first isolation and rest isolations are from subsequent supernatants. (Tables 1 and 2).
Total Volume of isolated RNA was 50 μl per isolation. The full RNA isolation protocol can be also obtained from manufacturer.
Measurement of RNA content with UV spectrometer, Nanodrop (Thermofischer, USA): The instrument was calibrated with elution buffer. After that, 2 ul of each isolated was measured and the values are recorded at μg/2 μl.
Real The results indicate that there is a loss of RNA during all steps and this loss decreases with the increase in the number of isolations. In the sample 2, it varies better 0.170 μg/2 μl at the beginning to 0.006 μg / 2 μl. It was very surprising to see that yield of isolated RNA was more in the first supernatant passage (second isolation) than the first isolation.
Real time PCR of isolated RNA in different isolated versions show that real time for beta actin housekeeping gene is in position to detect RNA in all isolations regardless whatever quantity was detected in spectrometer. Results are shown as curves in the Figure. It is hard to find any correlation between the amount of RNA measured in spectrometer and Ct values achieved in the real time machine.
Since the availability of possibility to isolate the nucleic acid with mini columns, it has become a common practice in all laboratories working with molecular methods, to conduct the RNA isolation from different samples to analyze them for different targets as these may be pathogenic agents and biomarkers of different cells e.g., stem cells, tumor cells, immune cells etc. It is also daily practice in the laboratories to measure the presence of viral RNA in different samples. [1,2,[11][12][13] Most of the laboratories are doing only once the isolation and throw away the samples as supernatant. Amount of isolated RNA with only one-time isolation is very limited e.g., 100 μl. If user is conducting experiments for a number of different targets, this small amount can be a limiting factor because of its small size. In the literature, there is hardly to find any report that some groups have tried to look into this important problem in spite of the fact that each group needs sufficient amount of nucleic acid to conduct the assays from the same source. This study is indicating that it is possible to isolate the more RNA from the supernatants of the same sample. In this way, many laboratories have sufficient amount of isolated RNA to conduct bigger studies and compare them also [14].
There are research groups, which are working on development of vaccines and therapies, they may face many times a challenge to isolate the sufficient RNA to conduct their experimental studies. This publication provides them the way to isolate more amount of valuable RNA rather throwing of the rest of supernatant.
These studies shown here can help many institutes and laboratories to save money in terms of chemicals and time used to isolate the samples. In two samples shown Table 2, the amounts of isolated RNA from sample number 3 and 4 were less than that of RNA isolated from two following supernatants from the first isolation. It should be investigated further as it indicates that there may be some time a lot of loss of RNA in the first isolation or binding capacity of membrane may be playing a role. User can have more isolated RNA in 2 nd and 3 rd subsequent isolations. There are many publications, where research groups are using 5 or 10 μl during the PCR studies, hence a lot of quantity of isolated RNA can be obtained from supernatant, which is usually thrown away as waste product. [15,16] This publication is showing a way to get larger volume of isolated RNA to bigger studies.
Fundin. This work is self-funded and there is no external funding.