Banana Slug Genomics

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======Banana Slug RNA======

=====RNA isolates=====

In Feburary 2015 the [[http://omegabiotek.com/store/product/e-z-n-a-mollusc-rna-kit/|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 [[http://www.lifetechnologies.com/order/catalog/product/AM1561|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 [[https://www.neb.com/products/n3231-100-bp-dna-ladder|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.


{{ :rnagel.png?nolink |}}


=====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 [[http://www.nanodrop.com/Productnd8000overview.aspx?gclid=CJaNntn0sMUCFU6VfgodXI4AlQ|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.
Banana Slug Genomics

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