RNA Isolation from Plant Tissues: a practical experience for

biological undergraduates

Manuel G. Claros, Francisco M. Canovas

Most of the plant material contains relatively high levels of RNAse activity mostly
located in the vacuole. During the RNA extraction in the plant it is necessary to completely break
the cells, minimize the activity of RNAses released during cell lysis and avoid introduction of the
minimum trace of the RNAses from any other source in the laboratory. This study focuses on the
extraction of the RNA from the spinach leaves and spinach stem. The students used various
techniques to extract the RNA from the subject that was used in the experiment.
The following procedure can be applied to a wide variety of plants invariably yielding
large amounts of undegraded, pure total RNA suitable for further analysis. Harvest5 g of
spinach leaves and grind them in liquid Nitrogen in a precooled mortar and pestle until a fine
powder is obtained.do not let the tissue thaw. Distribute 0.5 g of the powder to Eppendorf tubes
and add 250 microliter of TSB buffer and 250 microliter of APC. Stir and vortex until a complete
emulsion id formed. Spin for 2 minutes at 13000 rpm. Transfer the top aqueous phase to a new
prechilled tube. Add 500 micro liter of APC, mix vigorously and centrifuge for 2 minutes at
13000 rpm. Take the upper phase, add 500 microliter of chloroform, mix and centrifuge as
before. Use the upper phase. Hereafter RNA-grade materials must be used. Add 50 microliter of
3 M ammonium acetate and mix. Add 50 microliter of 2-propanol and precipitate on ice for 15
minutes. Centrifuge for 10 minutes at 13000 rpm at 4 degree Celsius. A white pellet should be
visible. Remove the supernatant and rinse with 500 microliter of 70% ethanol. Centrifuge for 2
minutes at 13000 rpm, remove the supernatant and dry under vacuum for 1-2 minutes. Dissolve
the pellet in 210 microliter of distilled water. If the suspension appears cloudy, spin at 13000
rpm for 5 minutes at 4 degree Celsius to remove insoluble particles. Carefully remove the
supernatant since the pellet may be compact. Then LiCLl precipitation is used to selectively
precipitate the RNA, removing the DNA and low molecular weight RNAs. Add 70 microliter of 8
M Lithium chloride and place on ice for 30 minutes. Centrifuge for 15 minutes at 13000 rpm at 4
degrees Celsius and carefully remove the supernatant. Rinse the pellet with 500 microliter of
70% ethanol and centrifuge for 2 minutes at 13000 rpm at 4 degrees Celsius. Remove the
supernatant a d dry by vacuum for 1-2 minutes. Dissolve the pellet in 100 microliter of distilled
water. The RNA can be stored at 70 Degrees Celsius. After that, prepare a dilution of 10
microliter of RNA in 490 microliter of distilled water and another 20 microliter in 480 microliter
distilled water. The absorbance at 310 nanometer should be zero to assure the absence of light
scattering which will otherwise influence further readings. Read the absorbance at 260 nm to
calculate het RNA concentration in both dilutions ( 1 absorbance unit at 160 nm = 40 mg/ml
RNA). Read the absorbance at 230 and 280 nm and calculate the ratios to check the purity.
 The study started the experiment or the methods in extracting the RNA from plants by
preparing a plant tissue homogenate in which the bulk of the RNA would be isolate. After
isolating huge amount of RNA, selective precipitation was done to choose which RNA should be
used in the study. In this process in the experiment, the researcher selected the RNA that the
study would be focusing on. After selecting which RNA, the concentration of the RNA was
computed by using spectrophotometer method and reading the absorbance of the sample at 260
nm. The results showed that the RNA obtained by the students had a concentration ranging from
1.1 to 2.9 mg/ml. The researcher can be deduced from the study that the RNA concentration was
not overestimated by any light scattering.
The results obtained from both of the spinach leaves and spinach stem had almost same
results; the main difference is that the one prepared from the stems contains less chloroplast
rRNA than that from the leaves. The method used by the students in this study are simple and
easy to carry out which can be also be used by other students who wish to do the same method in
their study. It also a great help to those students who want to learn how to calculate the
concentration of RNA in their study because this paper provide a method in which the students
can be used in calculating.