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Banana Slug RNA

RNA isolates

In Feburary 2015 the Omega Bio Tek E.Z.N.A.® Mollusc RNA kit was used to prepare 28 isolations of RNA from 16 tissue samples. Note that this kit is not recommended for RNA <200nt and there was no DNase treatment.

In April 2015 the mirVana™ miRNA Isolation Kit, without phenol was used to create a size selected sample of one of the RNA isolates (dg2). Later that month 9 RNA samples (V2, K, SMT, DA2, PA1, DG3, DG1, DG2-S, DG2-L) were run on a 1.5% CE Agarose gel compared to a NEB 100nt DNA ladder. Unfortunately there was a miscommunication between Jared and his supervisor it is mostly unknown which sample made it to what well. Note that there is still a presence of small RNA (<200 nt) but no distinct rRNA, tRNA, or miRNA bands.

If we decide to make small RNA libraries a different protocol will need to be run on one of the left-over tissue cuts.

RNA samples

Abbreviations for tissues cuts:

Tissue cut Abbreviation ID number of cuts from tissue
Penis p 3
Digestive Gland dg 3
Albumen gland (proximal) pa 2
Albumen gland (distal) da 2
Vagina v 2
Penis Muscle pm 2
Foot f 2
Salivary Gland sg 2
Ovotestis ot 2
Body Wall bw 2
Shell sh 1
Vas Deferons vd 1
Ovoduct od 1
Connective tissue ct 1
spermathecca smt 1
Kidney k 1

The following table of data was generated with a nanodrop

Sample ID Conc. (ng/ul) 260/280 260/230 Vol. (ul) Total (ug) notes
pa1 180.9 2 1.87 64.4 11.65
pa2 279.3 1.99 2.32 70 19.55
da1 150.2 2.04 1.36 70 10.51
da2 113.4 2.09 0.23 61 6.93 contaminated?
v1 112.0 2.02 0.97 70 7.84 tougher, lighter, bulbous
v2 78.42 2.03 0.83 56 4.39 flimsy, darker, sleeve-like
pm1 3.207 1.91 0.41 70 0.22
pm2 25.79 1.95 0.31 70 2.86
f1 40.84 1.88 0.89 70 2.86
f2 47.03 1.84 1.77 69 3.25
sg1 338.0 1.9 1.62 70 23.66 dense with dark veins
sg2 64.12 1.9 0.54 70 4.49 absent of dark veins
ot1 39.95 1.82 1.2 70 2.80
ot2 32.53 1.79 1.46 70 2.28
bw1 7.527 1.65 0.69 70 0.53
bw2 15.21 1.84 0.29 70 1.06
sh 3.768 1.23 0.39 70 0.26
vd 7.468 1.67 0.36 70 0.52
od 26.13 1.99 1.56 70 1.83
ct 40.43 1.92 1.82 70 2.83
smt 93.26 1.95 1.86 59 5.50 Steven spilled some
k 160.0 1.98 2.11 63.5 10.16
p1 53.08 1.89 1.59 70 3.72 only epiphallus
p2 16.91 1.75 1.06 70 1.18
p3 9.761 1.55 0.48 70 0.68 no epiphallus
dg1 439.6 1.95 2.1 67 29.45 iron?
dg2 253.6 1.98 2.16 0 0.00 size selected
dg3 429.4 1.99 2.12 67 28.77 iron?
dg2-S 91.24 2.07 1.83 98.5 8.99 <200nt
dg2-L 225.3 2.09 1.74 55.5 12.50 >200nt

260/280

The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. The five nucleotides that comprise DNA and RNA exhibit widely varying 260/280 ratios**. The following represent the ratios estimated for each nucleotide if measured independently:

  • Guanine: 1.15
  • Adenine: 4.50
  • Cytosine: 1.51
  • Uracil: 4.00
  • Thymine: 1.47

The resultant 260:280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260/280 ratios for the four nucleotides present. It is important to note that the generally accepted ratios of 1.8 and 2.0 for DNA and RNA respectively, are “rules of thumb”. The actual ratio will depend on the composition of the nucleic acid. Note: RNA will typically have a higher 260/280 ratio due to the higher ratio of Uracil compared to that of Thymine.

  • 260/280 ratio – a low ratio may be the result of a contaminant absorbing at 280 nm or less.
    • Protein contamination
    • Residual phenol or other reagent associated with the extraction protocol
    • A very low concentration( > 10 ng/ul).of nucleic acid

260/230

This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2.

  • 260/230 ratio – a low ratio may be the result of a contaminant absorbing at 230 nm or less.
    • Carbohydrate carryover (often a problem with plants).
    • Residual phenol from nucleic acid extraction.
    • Residual guanidine (often used in column based kits).
    • Glycogen used for precipitation.

Discussion

, 2015/05/12 09:42

I would like to flag this page for removal (or burial somewhere deep in the wiki), I don't see any use for the page when we don't have labeled lanes.

, 2015/05/22 13:33

just bury this page as old_RNA or something as you add new data here.

, 2015/05/08 08:38

How are we going to use these data when we don't know which lanes are which? Maybe I am misunderstanding this… “Unfortunately there was a miss-communication between Jared and his supervisor it is mostly unknown which sample made it to what well.”

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archive/rna.txt · Last modified: 2015/07/27 22:58 by ceisenhart